Neisseria meningitidis polypeptide, nucleic acid sequence and uses thereof

ABSTRACT

The invention discloses the  Neisseria meningitidis  NMASP polypeptide, polypeptides derived therefrom (NMASP-derived polypeptides), nucleotide sequences encoding said polypeptides, and antibodies that specifically bind the NMASP polypeptide and/or NMASP-derived polypeptides. Also disclosed are prophylactic or therapeutic compositions, including immunogenic compositions, e.g., vaccines, comprising NMASP polypeptide and/or a NMASP-derived polypeptide. The invention additionally discloses methods of inducing a immune response to  Neisseria meningitidis  and  Neisseria meningitidis  NMASP polypeptide and an NMASP-derived polypeptide in animals.

This application claims priority benefits of provisional U.S.application No. 60/098,685, filed Sep. 1, 1998, the entire disclosure ofwhich is incorporated by reference herein.

INTRODUCTION

The present invention relates generally to a polypeptide of Neisseriameningitidis of approximately 40-55 kD referred to as “NMASP”. Theinvention encompasses an isolated or purified NMASP polypeptide andpolypeptides, including fragments, derived therefrom (NMASP-derivedpolypeptides), and methods of making thereof. The invention alsoencompasses antibodies, including cytotoxic or bactericidal antibodies,that specifically bind the NMASP polypeptide, NMASP-derived polypeptidesand/or fragments thereof. The invention further encompasses immunogenic,prophylactic or therapeutic compositions, including vaccines, thatcomprise NMASP polypeptide, NMASP-derived polypeptides and/or fragmentsthereof. The invention additionally provides methods of inducing animmune response to Neisseria meningitidis in an animal and methods oftreating infections in an animal caused by Neisseria meningitidis Theinvention further provides isolated nucleotide sequences encoding theNMASP polypeptide, NMASP-derived polypeptides and fragments thereof,vectors having said sequences, and host cells containing said vectors.

BACKGROUND OF THE INVENTION

Neisseriae are gram-negative diplococci and include but are not limitedto Neisseria ovis, Neisseria lacunata, Neisseria osloensis, Neisseriabovis, Neisseria meningitidis, and Neisseria gonorrhoeae. Neisseriameningitidis (“N.m.”) is the most common cause of bacterial meningitidisand septicemia in infants and young adults in the industrialized world;markedly so in countries that have initiated immunization programsagainst Haemophilus influenzae type B (Hib) disease (Riedo, F. X. et al.1995. Epidemiology and prevention of meningococcal disease. Pediatr.Infect. Did. J. 14:643-657; Hart, C. A. And T. R. Rogers. 1993.Meningococcal disease. J. Med. Microbiol. 39:3-25; Jackson, L. A. And J.D. Wenger. 1993. Laboratory-based surveillance for meningococcal diseasein selected areas, United States, 1989-1991. MMWR 42:21-30). World-wide,N. meningitidis accounts for about ⅓ of all cases of bacterialmeningitis; with most countries showing an attack rate of >1/100,000population. Mortality as a whole is significantly higher with themeningococci than with Hib disease. Unlike Hib infections which arebasically sporadic limited outbreaks, epidemics of meningococcal diseaseoccur regularly throughout the world and cause great suffering anddeath. Attack rates during epidemics can exceed 600/100,000 (Hart, C. A.And T. R. Rogers. 1993. Meningococcal disease. J. Med. Microbiol.39:3-25; Jones, D. 1995. Epidemiology of meningococcal disease in Europeand the USA. In: Meningococcal Disease. Cartwright, K. (Ed.) WileyPress, New York, USA: 145-157). Despite the organism's sensitivity to awide variety of antibiotics and the impact antibiotic intervention hashad on the overall case fatality rate, meningococcal disease attackrates have changed very little since the introduction of antibacterialsand the fatality rate still remains between 7 and 15% even inindustrialized countries.

N.m. infection starts with colonization of the upper respiratory tract;primarily the tonsils and nasopharynx (Brandtzaeg, P. 1995. Pathogenesisof meningococcal infections. In: Meningococcal Disease. Cartwright, K.(Ed.), Wiley Press, New York, USA: 145-157). Once colonization isestablished, the organism can invade the underlying endothelium and gainentry into the circulatory system where it causes a rapid, fulminatemeningococcemia and/or progresses to the cerebrospinal fluid to cause anoften fatal meningitis. To reach the meninges, the organism mustinteract and circumvent two cellular barriers, the nasopharynx and theblood-brain barrier. Bacterial-host cell interactions are thus criticalfor the pathogenesis of N.m. Pili, cell surface attachment components,and the polysaccharide capsule all play essential roles in the initialattachment and colonization processes (Jerse, A. E. And R. F. Rest.1997. Adhesion and invasion by the pathogenic neisseria. TrendsMicrobiol.:217-221). Once colonization of the upper respiratory tracthas been achieved, the organism can down-regulate pili expression andcapsule synthesis and expresses other afimbrial adhesins and invasionproteins possibly masked by the capsule that allow the bacteria toinvade the underlying endothelial cells.

Based on the structural carbohydrate composition of the meningococcalcapsular polysaccharide (CPS), N.m. strains can be divided into a least12 serogroups, designated types A through L (Riedo, F. X. et al. 1995.Epidemiology and prevention of meningococcal disease. Pediatr. Infect.Did. J. 14:643-657; Hart, C. A. and T. R. Rogers. 1993. Meningococcaldisease. J. Med. Microbiol. 39:3-25). However, serogroups A, B, and Caccount for over 90% of meningococcal disease and are the serotypes mostoften associated with epidemic disease (Jones, D. 1995. Epidemiology ofmeningococcal disease in Europe and the USA. In: Meningococcal Disease.Cartwright, K. (Ed.) Wiley Press, New York, USA: 145-157). In the UnitedStates and most developed countries, roughly half of the meningococcalmeningitis cases are caused by serogroup B. The highest attack rates oftype B meningococcal disease are observed in young children under theage of two with the peak incidence seen in children less than 1 year ofage.

The CPS has been targeted as a prime vaccine candidate for themeningococci. Several laboratories have shown that anti-CPS antibodiespromote complement-mediated killing of organisms which belong to thesame but not different capsular serogroups (Gotschlich, E. C. et al.1977. The immune responses to bacterial polysaccharides in man. In:Antibodies in Human Diagnosis and Therapy. Haber, E. And R. M. Krause(Eds.), Raven Press, New York, USA: 391-402). The emergence ofsulfonamide-resistant organisms in military recruits spurred thedevelopment of CPS vaccines against serogroups A, C, and W. While thesevaccines are highly immunogenic and effective in adults, the immuneresponse elicited in infants is minimal and of short duration, dueprimarily to the fact that the very young respond poorly toT-cell-independent antigens like the CPS immunogen.

Prototype serogroup B polysaccharide vaccines have been produced butwere found to be poorly immunogenic in humans and gave rise to only lowavidity antibody that does not stimulate high levels ofcomplement-mediated killing or opsonization (Frasch, C. E. 1995.Meningococcal vaccines: past, present and future. In: MeningococcalDisease. Cartwright, K. (Ed.) Wiley Press, New York, USA: 145-157). Thepoor immunogenicity of the type B CPS is believed to result from thestructural similarity of the type B capsule polysaccharide to the sialicacid structures (_(—)−2,8 linkage) found on the surface of human brainneural cell glycoproteins (NCAMS) (Finne, J. et al. 1983. Occurrence ofalpha-2,8 linked polysialosyl units in neural cell adhesion molecules.Biochem. Biophys. Res. Comm. 112:482-487). The poor immuneresponsiveness of type B CPS and the possibility that anti-type Bcapsular antibody may recognize native human carbohydrate structures andpossibly trigger an autoimmune sequelae has resulted in a greateremphasis on the evaluation of alternative meningococcal surface antigensas potential vaccine candidates (Poolman, J. T., et al. 1986. Class ⅓outer membrane protein vaccine against group B, type 15, subtype 16meningococci. Dev. Biol. Stand. 63:147-152; Ala'Aldeen, D. A. A., et al,1994. Immune responses in humans and animals to meningococcaltransferrin-binding proteins: implications for vaccine design. Infect.Immun. 62:2984-2990; Gotschlich, E. C. 1991. The meningococcal serogroupB vaccine protection trials: concluding remarks at the report meetingsecond day. NIPH Ann. 14:247-250; Noronha, C. P., et al., 1995.Assessment of the direct effectiveness of BC meningococcal vaccine inRio de Janerio, Brazil: a case-control study. Int. J. Epidemiol.24:1050-1057; Boslego, J. W. Et al. 1995. Efficacy, safety, andimmunogenicity of a meningococcal group B(15:P1.3) outer membraneprotein vaccine in Iquique Chile. Chilean National Committee forMeningococcal Disease. Vaccine. 13:821-829).

Outer membrane complexes as well as individual outer membranecomponents, including lipids, phospholipids, lipopolysaccharides andproteins, have been evaluated as potential N.m. B vaccines (Dalseg, R.,et al., 1995. Group B meningococcal OMV vaccine as a mucosal immunogen.Clin. Immunol. Immunopathol. 76:S93; Hoiby, E. A., et al., 1991.Bacteriocidal antibodies after vaccination with the Norwegianmeningococcal serogroup B outer membrane vesicle vaccine: a briefsurvey. NIPH Ann. 14:147-156; Jarvis, G. A., and J. M. Griffiss. 1991.Human IgA1 blockage of IgG-initiated lysis of N.m. is a function ofantigen-binding fragment binding to the polysaccharide capsule. J.Immunol. 147:1962-1967). While outer membrane bleb-based and outermembrane vesicle-based (OMVs) vaccines are able to elicit at least somedegree of bactericidal antibodies and mild cross-strain protection inyoung children, these vaccines are difficult and problematic to preparewhich renders them impractical as commercial vaccines.

The class I and class II outer membrane porin proteins (PorA, PorB), theiron-inducible transferrin/lactoferrin-binding proteins, the class Vopacity adhesin(s), and the class I/II surface fimbrial adhesins (pili)have been suggested as possible subunit vaccine candidates. Variousinvestigators have shown that although all these proteins areimmunogenic and some even elicit bacteriocidal activity, they all show avery high degree of antigenic variability. The surface-exposedstrain-variable domains of these proteins also correspond toneutralizing B-cell epitopes (Poolman, J. T. 1995. Surface structure andsecreted products of meningococci. In: Meningococcal Disease.Cartwright, K. (Ed.) Wiley Press, New York, USA: 145-157). Due to theantigenic variation among the major outer membrane proteins of themeningococci, these proteins confer limited cross-strain protection andare thus not suitable as cross-protective subunit vaccines. Thus, aneffective cross-protective N.m. type B subunit vaccine candidate must behighly conserved as well as immunogenic.

The HtrA protein has been identified as a virulence factor for severalbacterial pathogens including, Yersinia enterocolitica, Brucellaabortus, and Salmonella typhimurium. In some but not all organisms HtrAappears to be a stress-responsive protein, possibly contributing to theorganisms survival under oxidative challenge and/or at elevatedtemperatures. The exact role HtrA plays during the pathogenesis processhas not yet been fully defined. Bacteria-host cell interaction and theresulting signal transduction events that are triggered in the pathogenmay promote expression of the HtrA protein. The E. coli and H.influenzae HtrA proteins, including the Hin47 protein described in U.S.Pat. Nos. 5,679,547 and 5,721,115, both of which are incorporated hereinby reference in their entireties, have been shown to be serine proteasesand possess three relatively conserved domains that house the catalyticresidues H, D and S.

HtrA is a virulence factor, having serine protease activity, which hasrecently been identified as a target for the development ofanti-bacterial agents against gram negative bacterial pathogens. (Jonesand Hruby, 1998, New targets for antibiotic development: biogenesis ofsurface adherence structures, DDT Vol.3(11)495-504; Barrett and Hoch,1998, Two-component signal transduction as a target for microbialanti-infective therapy, Antimicrobial. Agents and Chemother.42(7):1529-1536; Fabret and Hoch, 1998, A two-component signaltransduction system essential for growth of Bacillus subtilis:implications for anti-infective therapy, J. Bacteriol.,180(23):6375-6382).

Citation or identification of any reference in this section or any othersection of this application shall not be construed as an indication thatsuch reference is available as prior art to the present invention.

SUMMARY OF THE INVENTION

One object of this invention is to identify and provide a novel andhighly conserved protein (referred to hereafter and in the claims as“NMASP”) from Neisseria meningitidis. The protein of the presentinvention has a molecular weight of approximately 40-55 kD, and haslimited similarity (˜36% identity) BLAST Program (Altschul et al., 1990,J. Molec. Biol. 215:403-10; Altschul et al., 1997, Nuc. Acids Res.25:3389-3402) with data entered using FASTA format; expect 10 filterdefault; description 100, alignment[overall), to the DegP (HtrA) proteinof E. coli and has not been previously identified in any Neisseriameningitidis. The protein sequence which is another object of thisinvention has similarity to several DegP/HtrA-like seine proteases fromtwo other bacteria and these sequence homologies have not beenpreviously reported for any Neisseria meningitidis.

The invention is based, in part, on the surprising discovery thatNeisseria meningitidis, and various strains and cultivars thereof, havea protein, NMASP polypeptide, which is about 40 kD to about 55 kD inmolecular weight, preferably about 44 kD to about 53 kD.

The present invention encompasses the NMASP polypeptide of Neisseriameningitidis in isolated or recombinant form. The invention encompassesa purified NMASP polypeptide, polypeptides derived therefrom(NMASP-derived polypeptides), and methods for making said polypeptideand derived polypeptides. The invention also encompasses antisera andantibodies, including cytotoxic or bactericidal antibodies, which bindto and are specific for the NMASP polypeptide, NMASP-derivedpolypeptides and/or fragments thereof.

The invention further encompasses pharmaceutical compositions includingprophylactic or therapeutic compositions and which may be antigenic orimmunogenic compositions including vaccines, comprising one or more ofsaid polypeptides, optionally in combination with, fused to orconjugated to another component, including a lipid, phospholipid, acarbohydrate including a lipopolysaccharide or any of the proteins,particularly any Neisseria, Moraxella, Pseudomonas, Streptococcus orHaemophilus protein known to those skilled in the art. The inventionfurther encompasses pharmaceutical compositions including prophylacticor therapeutic compositions, which may be antigenic, preferablyimmunogenic compositions including vaccines, comprising one or more ofthe NMASP polypeptide and NMASP-derived polypeptides and an attenuatedor inactivated Neisseria, Moraxella, Pseudomonas, Streptococcus orHaemophilus cultivar or an attenuated or inactivated Neisseria cultivarexpressing NMASP polypeptide in a greater amount when compared towild-type Neisseria.

The invention additionally provides methods of inducing an immuneresponse to Neisseria meningitidis in an animal and methods of treatingor preventing an infection caused by Neisseria meningitidis in ananimal.

The invention further provides isolated nucleotide sequences encodingthe NMASP polypeptide, NMASP-derived polypeptides, and fragmentsthereof, vectors having said sequences, host cells containing saidvectors, recombinant polypeptides produced therefrom, and pharmaceuticalcompositions comprising the nucleotide sequences, vectors, and cells.The nucleotide sequence of the NMASP nucleic acid is shown in SEQ IDNO:1. A deduced amino acid sequence of the open reading frame of NMASPis shown in SEQ ID NO:2.

In other embodiments of the invention there are provided methods foridentifying compounds which bind to or otherwise interact with andinhibit or activate an activity of a NMASP peptide or polypeptide or theDNA sequences of the invention encoding same comprising: contacting theDNA or polypeptide to assess the binding or other interaction, suchbinding or interaction being associated with a binding or interaction ofthe DNA or polypeptide with the compound and determining whether thecompound binds to or otherwise interacts with and activates or inhibitsan activity of the DNA or polypeptide by detecting the presence orabsence of a signal generated from the binding or interaction of thecompound with the DNA or polypeptide. In accordance with another aspectof the invention, there are provided NMASP agonist or antagonists,preferably bacteriostatic bacteriocidal agonists or antagonists.

One advantage of this invention is that antibody generated against thenewly discovered NMASP polypeptide of the present invention, in ananimal host will exhibit bactericidal and/or opsonic activity againstmany Neisseriae meningitidis strains and thus confer broad cross-strainprotection. Bactericidal and/or opsonic antibody will prevent thebacterium from infecting the host and/or enhance the clearance of thepathogen by the host's immune system. Neisseria meningitidis antibodybactericidal activity is the principal laboratory test that has beencorrelated with protection in humans and is the standard assay in thefield as being predictive of a vaccine's efficacy against Neisseriameningitidis infections. Bactericidal antibodies are particularlyimportant for N.m. vaccines because there is no natural animal hostother than humans and thus there is no relevant predictive animal modelof disease.

3.1. DEFINITIONS AND ABBREVIATIONS anti-NMASP = a polyclonal ormonoclonal antibody or antiserum that binds to a NMASP polypeptide orNMASP-derived polypeptide ATCC = American Type Culture Collection blebs= naturally occurring outer membrane vesicles of Neisseria meningitidisantigenic = capable of binding specifically to antibody or T cellreceptors and provoking an immune response immunogenic = capable ofprovoking a protective cellular or humoral immune response kD =kilodaltons N. = Neisseria NMASP = a non-cytosolic polypeptide of aNeisseria meningitidis, or any strain or cultivar thereof, having amolecular weight of about 40 kD to 55 kD; NMASP-derived polypeptide =fragment of the NMASP polypeptide; variant of wild-type NMASPpolypeptide or fragment thereof, containing one or more amino aciddeletions, insertions or substitutions; or chimeric protein comprising aheterologous polypeptide fused to a C-terminal or N-terminal or internalsegment of a whole or a portion of the NMASP polypeptide; OG =n-octyl-β-D-glucopyranoside or octylglucoside PBS = phosphate bufferedsaline PAG = polyacrylamide gel polypeptide = a peptide or protein ofany length, preferably one having eight or more amino acid residues SDS= sodium dodecylsulfate SDS-PAGE = sodium dodecylsulfate polyacrylamidegel electrophoresis

Nucleotide or nucleic acid sequences defined herein are represented byone-letter symbols for the bases as follows:

A (adenine)

C (cytosine)

G (guanine)

T (thymine)

U (uracil)

M (A or C)

R(A or G)

W (A or T/U)

S (C or G)

Y (C or T/U)

K (G or T/U)

V (A or C or G; not T/U)

H (A or C or T/U; not G)

D (A or G or T/U; not C)

B (C or G or T/U; not A)

N (A or C or G or T/U) or (unknown)

Peptide and polypeptide sequences defined herein are represented byone-letter or three symbols for amino acid residues as follows:

1 letter 3 letter amino acid A Ala (alanine) R Arg (arginine) N Asn(asparagine) D Asp (aspartic acid) C Cys (cysteine) Q Gln (glutamine) EGlu (glutamic acid) G Gly (glycine) H His (histidine) I Ile (isoleucine)L Leu (leucine) K Lys (lysine) M Met (methionine) F Phe (phenylalanine)P Pro (proline) S Ser (serine) T Thr (threonine) W Trp (tryptophan) YTyr (tyrosine) V Val (valine) X Xaa (unknown)

The present invention may be more fully understood by reference to thefollowing detailed description of the invention, non-limiting examplesof specific embodiments of the invention and the appended figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Map of NMASP vector pNmAH116.

FIG. 2: NMASP protein expressed from TOP10(pNmAH145), uninduced (SDS:lane 2, Western blot: lane 4) and IPTG induced (SDS: lane 3, Westernblot: lane 5). A monoclonal anti-(His)₅ antibody conjugated to HRP(QiaGen) was used to identify the protein and visualization of theantibody reactive pattern was achieved on Hyperfilm using the AmershamECL chemiluminescence system. Lane 1 shows Novex MultiMark molecularweight markers of myosin (250 kD), phosphorylase B (148 kD), glutamicdehydrogenase (62 kD), carbonic anhydrase (42 kD), myoglobin-blue (30kD), myoglobin-red (22 kD), and lysozyme (17 kD).

DETAILED DESCRIPTION OF THE INVENTION NMASP Polypeptide

The invention provides an isolated or a substantially pure native (wildtype) or recombinantly produced polypeptide, referred to as NMASP, ofNeisseria meningitidis, and various strains or cultivars thereof. TheNMASP polypeptide comprises the whole or a subunit of a non-cytosolicprotein embedded in, or located in the bacterial envelope, which mayinclude the inner membrane, outer surface, and periplasmic space. TheNMASP polypeptide has an apparent molecular weight, as determined fromthe deduced amino acid sequence, of about 40 kD to about 55 kD,preferably about 44 kD to about 53 kD.

NMASP polypeptide may also be identified as the polypeptide inhydrophobic (salt) or detergent extracts of Neisseria meningitidis blebsor intact cells that has an apparent molecular weight about 40 kD toabout 55 kD, preferably about 44 kD to about 53 kD, as determined bydenaturing gel electrophoresis in 12% PAG with SDS, using formulationsas described in Harlow and Lane (Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Appendix I,1988).

In particular embodiments, the NMASP polypeptide is that obtainable fromany of Neisseria meningitidis, including, but not limited to types A-Land W. Preferred are N.m. Type A, Type B, Type C and Type W. Strainsfrom any of these organisms may be obtained worldwide from anybiologicals depository, particularly strains of Nm. Type A: ATCC13077,ATCC53417; Type B ATCC13090, ATCC13091, ATCC13092, ATCC13093, ATCC13094,ATCC13096, ATCC13098, ATCC13100, ATCC23247, ATCC23249, ATCC23250,ATCC23251, ATCC23253, ATCC23254, ATCC23255, ATCC23583, ATCC33086,ATCC53044, ATCC53415, ATCC53418; Type C ATCC13102, ATCC13103, ATCC13105,ATCC13106, ATCC132107, ATCC13108, ATCC13109, ATCC13110, ATCC13111,ATCC13112, ATCC23252, ATCC23248, ATCC31275, ATCC53414, ATCC53416,ATCC53900; and Type 29-E ATCC35558.

In a particular embodiment, the NMASP polypeptide comprises a deducedamino acid sequence as depicted in SEQ ID NOs: 2, 11 or 12. Particularlypreferred fragments of NMASP have deduced amino acid sequences depictedin SEQ ID NOs: 5-7, and 16. In another particular embodiment, the NMASPpolypeptide is encoded by the nucleotide sequence of SEQ ID NOs: 1, 10or 13, with particularly preferred fragments encoded by nucleotidesequences depicted in SEQ ID NOs: 3, 4, 8, 9, 14, 15, and 17-20. Inanother embodiment, the NMASP polypeptide comprises an amino acidsequence which is substantially homologous to the deduced amino acidsequence of SEQ ID NOs: 2, 11 or 12 or a portion thereof or is encodedby a nucleotide sequence substantially homologous to the nucleotidesequence of SEQ ID No: 1, 10 or 13 or a portion thereof.

As used herein a “substantially homologous” sequence is at least 70%,preferably greater than 80%, more preferably greater than 90% identicalto a reference sequence of identical size or when compared to areference sequence when the alignment or comparison is conducted by acomputer homology program or search algorithm known in the art. By wayof example and not limitation, useful computer homology programs includethe following: Basic Local Alignment Search Tool (BLAST) (Altschul etal., 1990, J. of Molec. Biol., 215:403-410, “The BLAST Algorithm;Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402) a heuristic searchalgorithm tailored to searching for sequence similarity which ascribessignificance using the statistical methods of Karlin and Altschul 1990,Proc. Nat'l Acad. Sci. USA, 87:2264-68; 1993, Proc. Nat'l Acad. Sci. USA90:5873-77. Five specific BLAST programs perform the following tasks:

1) The BLASTP program compares an amino acid query sequence against aprotein sequence database.

2) The BLASTN program compares a nucleotide query sequence against anucleotide sequence database.

3) The BLASTX program compares the six-frame conceptual translationproducts of a nucleotide query sequence (both strands) against a proteinsequence database.

4) The TBLASTN program compares a protein query sequence against anucleotide sequence database translated in all six reading frames (bothstrands).

5) The TBLASTX program compares the six-frame translations of anucleotide query sequence against the six-frame translations of anucleotide sequence database.

Smith-Waterman (database: European Bioinformatics Institute(Smith-Waterman, 1981, J. of Molec. Biol., 147:195-197) is amathematically rigorous algorithm for sequence alignments.

FASTA (see Pearson et al., 1988, Proc. Nat'l Acad. Sci. USA,85:2444-2448) is a heuristic approximation to the Smith-Watermanalgorithm. For a general discussion of the procedure and benefits of theBLAST, Smith-Waterman and FASTA algorithms see Nicholas et al., 1998, “ATutorial on Searching Sequence Databases and Sequence Scoring Methods”and references cited therein.

By further way of example and not limitation, useful computer homologyalgorithms and parameters for determining percent identity include thefollowing:

To determine the percent identity of two amino acid sequences or of twonucleic acids, e.g. between Thy-1 sequences and other known sequences,the sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in the sequence of a first amino acid or nucleic acidsequence for optimal alignment with a second amino or nucleic acidsequence). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position. The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences (i.e., % identity=# ofidentical positions/total # of positions (e.g., overlappingpositions)×100). In one embodiment, the two sequences are the samelength.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul, 1993,Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm isincorporated into the NBLAST and XBLAST programs of Altschul, et al.,1990, J. Mol. Biol. 2 15:403-410. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to a nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to a protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402.Alternatively, PSI-Blast can be used to perform an iterated search whichdetects distant relationships between molecules (Id.). When utilizingBLAST, Gapped BLAST, and PSI-Blast programs, the default parameters ofthe respective programs (e.g., XBLAST and NBLAST) can be used. Anotherpreferred, non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the CGC sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used. Additional algorithms for sequenceanalysis are known in the art and include ADVANCE and ADAM as describedin Torellis and Robotti (1994) Comput. Appl. Biosci., 10:3-5; and FASTAdescribed in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-8.Within FASTA, ktup is a control option that sets the sensitivity andspeed of the search. If ktup=2, similar regions in the two sequencesbeing compared are found by looking at pairs of aligned residues; ifktup=1, single aligned amino acids are examined. ktup can be set to 2 or1 for protein sequences, or from 1 to 6 for DNA sequences. The defaultif ktup is not specified is 2 for proteins and 6 for DNA.

Alternatively, protein sequence alignment may be carried out using theCLUSTAL W algorithm, as described by Higgins et al., 1996, MethodsEnzymol. 266:383-402.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, only exact matches are counted.

According to various aspects of the invention, the polypeptides of theinvention are characterized by their apparent molecular weights based onthe polypeptides' migration in SDS-PAGE relative to the migration ofknown molecular weight markers. While any molecular weight standardsknown in the art may be used with the SDS-PAGE, preferred molecularweight markers comprise at least glutamic dehydrogenase and carbonicanhydrase. Other molecular weight markers include bovine serum albumin,chicken ovalbumin, bovine carbonic anhydrase. One skilled in the artwill appreciate that the polypeptides of the invention may migratedifferently in different types of gel systems (e.g., different buffers;different types and concentrations of gel, crosslinkers or SDS, etc.).One skilled in the art will also appreciate that the polypeptides mayhave different apparent molecular weights due to different molecularweight markers used with the SDS-PAGE. Hence, the molecular weightcharacterization of the polypeptides of the invention is intended to bedirected to cover the same polypeptides on any SDS-PAGE systems and withany molecular weight markers which might indicate sightly differentapparent molecular weights for the polypeptides than those disclosedherein.

NMASP-derived Polypeptides

An NMASP-derived polypeptide of the invention may be a fragment of theNMASP polypeptide. Fragments include those polypeptides having 7 or moreamino acids; preferably 8 or more amino acids; more preferably 9 or moreamino acids; and most preferably 10 or more amino acids of the NMASPpolypeptide.

The intact NMASP polypeptide may contain one or more amino acid residuesthat are not necessary to its immunogenicity. It may be the case, forexample, that only the amino acid residues forming a particular epitopeof the NMASP polypeptide are necessary for immunogenic activity.Unnecessary amino acid sequences can be removed or modified bytechniques well known in the art, i.e., an NMASP-derived polypeptide.

Preferably, the NMASP-derived polypeptides of the invention areantigenic, i.e. binding specifically to an anti-NMASP antibody and morepreferably the NMASP-derived polypeptides are immunogenic andimmunologically cross-reactive with the NMASP polypeptide, thus beingcapable of eliciting in an animal an immune response to Neisseriameningitidis. More preferably, the NMASP-derived polypeptides of theinvention comprise sequences forming one or more epitopes of the nativeNMASP polypeptide of Neisseria meningitidis (i.e., the epitopes of NMASPpolypeptide as it exists in intact Neisseria meningitidis cells). Suchpreferred NMASP-derived polypeptides can be identified by their abilityto specifically bind antibodies raised to intact Neisseria meningitidiscells (e.g., antibodies elicited by formaldehyde or glutaraldehyde fixedNeisseria meningitidis cells; such antibodies are referred to herein as“anti-whole cell” antibodies). For example, polypeptides or peptidesfrom a limited or complete protease digestion of the NMASP polypeptideare fractionated using standard methods and tested for their ability tobind anti-whole cell antibodies. Reactive polypeptides comprisepreferred NMASP-derived polypeptides. They are isolated and their aminoacid sequences determined by methods known in the art.

Also preferably, the NMASP-derived polypeptides of the inventioncomprise sequences that form one or more epitopes of native NMASPpolypeptide that mediate bactericidal or opsonizing antibodies. Suchpreferred NMASP-derived polypeptides may be identified by their abilityto generate antibodies that kill Neisseria meningitidis cells. Forexample, polypeptides from a limited or complete protease digestion orchemical cleavage of NMASP polypeptide are fractionated using standardmethods, injected into animals and the antibodies produced therefromtested for the ability to interfere with or kill Neisseria meningitidiscells. Once identified and isolated, the amino acid sequences of suchpreferred NMASP-derived polypeptides are determined using standardsequencing methods. The determined sequence may be used to enableproduction of such polypeptides by synthetic chemical and/or geneticengineering means.

These preferred NMASP-derived polypeptides also can be identified byusing anti-whole cell antibodies to screen bacterial librariesexpressing random fragments of Neisseria meningitidis genomic DNA orcloned nucleotide sequences encoding the whole NMASP polypeptide orfragments thereof. See, e.g., Sambrook et al., Molecular Cloning, ALaboratory Manual, 2nd ed., Cold Spring Harbor Press, New York, Vol. 1,Chapter 12. The reactive clones are identified and their inserts areisolated and sequenced to determine the amino acid sequences of suchpreferred NMASP-derived polypeptides.

By way of example and not limitation, the unwanted amino acid sequencescan be removed by limited proteolytic digestion using enzymes such astrypsin, papain, or related proteolytic enzymes or by chemical cleavageusing agents such as cyanogen bromide and followed by fractionation ofthe digestion or cleavage products.

An NMASP-derived polypeptide of the invention may also be a modifiedNMASP polypeptide or fragment thereof (i.e., an NMASP polypeptide orfragment having one or more amino acid substitutions, insertions and/ordeletions of the wild-type NMASP sequence or amino acids chemicallymodified in vivo or in vitro). Such modifications may enhance theimmunogenicity of the resultant polypeptide product or have no effect onsuch activity. Modification techniques that may be used include thosedisclosed in U.S. Pat. No. 4,526,716.

As an illustrative, non-limiting example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity which acts as a functional equivalent, resulting in asilent alteration. Substitutes for an amino acid within the sequence maybe selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. The polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The positivelycharged (basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid.

An NMASP-derived polypeptide of the invention may also be a moleculecomprising a region that is substantially homologous to (e.g., invarious embodiments, at least 60% or 70% or 80% or 90% or 95% identityover an amino acid sequence of identical size or when compared to analigned sequence in which the alignment is performed by a computerhomology program known in the art) or whose encoding nucleic acid iscapable of hybridizing to a coding NMASP sequence, under highlystringent, moderately stringent, or low or nonstringent conditions.

By way of example and not limitation, useful computer homology programsinclude the following: Basic Local Alignment Search Tool (BLAST)(Altschul et al., 1990, J. of Molec. Bid., 215:403-410, “The BLASTAlgorithm; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402) aheuristic search algorithm tailored to searching for sequence similaritywhich ascribes significance using the statistical methods of Karlin andAltschul (1990, Proc. Nat'l Acad. Sci. USA, 87:2264-68; 1993, Proc.Nat'l Acad. Sci. USA 90:5873-77). Two specific BLAST programs performthe following tasks:

1) The BLASTP program compares an amino acid query sequence against aprotein sequence database; and

2) The BLASTN program compares a nucleotide query sequence against anucleotide sequence database; and hence are useful to identify,respective substantially homologous amino acid and nucleotide sequences.

Additional algorithms which can be useful are the Smith-Waterman andFASTA algorithms. See supra Section 5.1 for a more detailed descriptionof useful algorithms and parameters for determining percent identity ofnucleotide (and/or amino acid) sequences.

Included within the scope of the invention are NMASP-derivedpolypeptides which are NMASP polypeptide fragments or other derivativesor analogs which are differentially modified during or aftertranslation, e.g., by glycosylation, acetylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to an antibody molecule or other cellularligand, etc. Any of numerous chemical modifications may be carried outby known techniques, including but not limited to specific chemicalcleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the NMASPpolypeptide sequence. Non-classical amino acids include but are notlimited to the D-isomers of the common amino acids, α-amino isobutyricacid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine,citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acidssuch as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

An NMASP-derived polypeptide may further be a chimeric polypeptidecomprising one or more heterologous polypeptides, lipids, phospholipidsor lipopolysaccharides of Neisserial origin or of another bacterialorigin, fused to the amino-terminal or carboxyl-terminal or internal ofa complete NMASP polypeptide or a portion of or a fragment thereof.Useful heterologous polypeptides comprising such chimeric polypeptideinclude, but are not limited to, a) pre- and/or pro-sequences thatfacilitate the transport, translocation and/or processing of theNMASP-derived polypeptide in a host cell, b) affinity purificationsequences, and c) any useful immunogenic sequences (e.g., sequencesencoding one or more epitopes of a surface-exposed protein of amicrobial pathogen). One preferred heterologous protein of the chimericpolypeptide includes Hin47 (see U.S. Pat. Nos. 5,679,547 and 5,721,115).

Isolation and Purification of NMASP Polypeptide and NMASP-derivedPolypeptides

The invention provides isolated NMASP polypeptides and NMASP-derivedpolypeptides. As used herein, the term “isolated” means that the productis significantly free of other biological materials with which it isnaturally associated. That is, for example, an isolated NMASPpolypeptide composition is between about 70% and 94% pure NMASPpolypeptide by weight. Preferably, the NMASP polypeptides andNMASP-derived polypeptides of the invention are purified. As usedherein, the term “purified” means that the product is substantially freeof other biological material with which it is naturally associated. Thatis, a purified NMASP polypeptide composition is at least 95% pure NMASPpolypeptide by weight, preferably at least 98% pure NMASP polypeptide byweight, and most preferably at least 99% pure NMASP polypeptide byweight.

The NMASP polypeptide of the invention may be isolated from a proteinextract including a whole cell extract, of any Neisseria meningitidis,including, but not limited to, types A-L and W. Preferred are N.m. TypeA, Type B, Type C and Type W. Strains from any of these organisms may beobtained worldwide from any biologicals depository, particularly strainsof N.m. Type A: ATCC13077, ATCC53417; Type B ATCC13090, ATCC13091,ATCC13092, ATCC13093, ATCC13094, ATCC13096, ATCC13098, ATCC13100,ATCC23247, ATCC23249, ATCC23250, ATCC23251, ATCC23253, ATCC23254,ATCC23255, ATCC23583, ATCC33086, ATCC53044, ATCC53415, ATCC53418; Type CATCC13102, ATCC13103, ATCC13105, ATCC13106, ATCC132107, ATCC13108,ATCC13109, ATCC13110, ATCC13111, ATCC13112, ATCC23252, ATCC23248,ATCC31275, ATCC53414, ATCC53416, ATCC53900; and Type 29-E ATCC35558.Another source of the NMASP polypeptide is a protein preparation from agene expression system expressing a sequence encoding NMASP polypeptideor NMASP-derived polypeptides (see Section 5.7., infra).

The NMASP polypeptide can be isolated and purified from the sourcematerial using any biochemical technique and approach well known tothose skilled in the art. In one approach, Neisseria cellular envelopeis obtained by standard techniques and inner membrane, periplasmic andouter membrane proteins are solubilized using a solubilizing agent suchas a detergent or hypotonic solution. A preferred detergent solution isone containing octyl glucopyranoside (OG), sarkosyl or TRITON X100™(t-Octylphenoxy polyethoxyethanol). A preferred solubilizing hypotonicsolution is one containing LiCl. NMASP polypeptide is in the solubilizedfraction. Cellular debris and insoluble material in the extract areseparated and removed preferably by centrifuging. The polypeptides inthe extract are concentrated, incubated in SDS-containing Laemmli gelsample buffer at 100° C. for 5 minutes and then fractionated byelectrophoresis in a denaturing sodium dodecylsulfate (SDS)polyacrylamide gel (PAG) from about 6% to about 12%, with or without areducing agent. See Laemmli, 1970, Nature 227:680-685. The band orfraction identified as NMASP polypeptide, having an apparent molecularweight of about 40 kD to about 55 kD, as described above, may then beisolated directly from the fraction or gel slice containing the NMASPpolypeptide. In a preferred embodiment, NMASP polypeptide has anapparent molecular weight of about 44 kD to about 53 kD which could bedetermined by comparing its migration distance or rate in a denaturingSDS-PAGE relative to those of bovine serum albumin (66.2 kD) and chickenovalbumin (45 kD).

Another method of purifying NMASP polypeptide is by affinitychromatography using anti-NMASP antibodies, (see Section 5.5).Preferably, monoclonal anti-NMASP antibodies are used. The antibodiesare covalently linked to agarose gels activated by cyanogen bromide orsuccinimide esters (Affi-Gel, BioRad, Inc.) or by other methods known tothose skilled in the art. The protein extract is loaded on the top ofthe gel as described above. The contact is for a period of time andunder standard reaction conditions sufficient for NMASP polypeptide tobind to the antibody. Preferably, the solid support is a material usedin a chromatographic column. NMASP polypeptide is then removed from theantibody, thereby permitting the recovery NMASP polypeptide in isolated,or preferably, purified form.

An NMASP-derived polypeptide of the invention can be produced bychemical and/or enzymatic cleavage or degradation of isolated orpurified NMASP polypeptide. An NMASP-derived polypeptide can also bechemically synthesized based on the known amino acid sequence of NMASPpolypeptide and, in the case of a chimeric polypeptide, the amino acidsequence of the heterologous polypeptide by methods well known in theart. See, for example, Creighton, 1983, Proteins: Structures andMolecular Principles, W. H. Freeman and Co., New York.

An NMASP-derived polypeptide can also be produced in a gene expressionsystem expressing a recombinant nucleotide construct comprising asequence encoding NMASP-derived polypeptides. The nucleotide sequencesencoding polypeptides of the invention may be synthesized, and/orcloned, and expressed according to techniques well known to thoseskilled in the art. See, for example, Sambrook, et al., 1989, MolecularCloning, A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Press, NewYork, Chapter 9.

NMASP-derived polypeptides of the invention can be fractionated andpurified by the application of standard protein purification techniques,modified and applied in accordance with the discoveries and teachingsdescribed herein. In particular, preferred NMASP-polypeptides of theinvention, those that form an outer-surface or exposed epitope of thenative NMASP polypeptide may be isolated and purified according to theaffinity procedures disclosed above for the isolation and purificationof NMASP polypeptide (e.g., affinity purification using anti-NMASPantibodies).

If desirable, the polypeptides of the invention may be further purifiedusing standard protein or peptide purification techniques including butnot limited to electrophoresis, centrifugation, gel filtration,precipitation, dialysis, chromatography (including ion exchangechromatography, affinity chromatography, immunoadsorbent affinitychromatography, reverse-phase high performance liquid chromatography,and gel permeation high performance liquid chromatography), isoelectricfocusing, and variations and combinations thereof.

One or more of these techniques may be employed sequentially in aprocedure designed to isolate and/or purify the NMASP polypeptide or theNMAP-derived polypeptides of the invention according to its/theirphysical or chemical characteristics. These characteristics include thehydrophobicity, charge, binding capability, and molecular weight of theprotein. The various fractions of materials obtained after eachtechnique are tested for their abilities to bind the NMASP receptor orligand, to bind anti-NMASP antibodies or to have serine proteaseactivity (“test” activities). Those fractions showing such activity arethen subjected to the next technique in the sequential procedure, andthe new fractions are tested again. The process is repeated until onlyone fraction having the above described “test” activities remains andthat fraction produces only a single band or entity when subjected topolyacrylamide gel electrophoresis or chromatography.

NMASP Immunogens and Anti-NMASP Antibodies

The present invention provides antibodies that specifically bind NMASPpolypeptide or NMASP-derived polypeptides. For the production of suchantibodies, isolated or preferably, purified preparations of NMASPpolypeptide or NMASP-derived polypeptides are used as antigens in anantigenic composition, more preferably as immunogens in an immunogeniccomposition.

In an embodiment, the NMASP polypeptide is separated from other outermembrane or periplasmic proteins present in the extracts of Neisseriameningitidis cells or blebs using SDS-PAGE (see Section 5.3. above) andthe gel slice containing NMASP polypeptide is used as an immunogen andinjected into a rabbit to produce antisera containing polyclonal NMASPantibodies. The same immunogen can be used to immunize mice for theproduction of hybridoma lines that produce monoclonal anti-NMASPantibodies. In particular embodiments, the immunogen is a PAG slicecontaining isolated or purified NMASP from any Neisseria meningitidis,including, but not limited to, types A-L and W. Preferred are N.m. TypeA, Type B, Type C and Type W. Particularly preferred are the strains ofN.m. Type A: ATCC13077, ATCC53417; Type B ATCC13090, ATCC13091,ATCC13092, ATCC13093, ATCC13094, ATCC13096, ATCC13098, ATCC13100,ATCC23247, ATCC23249, ATCC23250, ATCC23251, ATCC23253, ATCC23254,ATCC23255, ATCC23583, ATCC33086, ATCC53044, ATCC53415, ATCC53418; Type CATCC13102, ATCC13103, ATCC13105, ATCC13106, ATCC132107, ATCC13108,ATCC13109, ATCC13110, ATCC13111, ATCC13112, ATCC23252, ATCC23248,ATCC31275, ATCC53414, ATCC53416, ATCC53900; and Type 29-E ATCC35558.

In other embodiments, peptide fragments of NMASP polypeptide are used asimmunogens. Preferably, peptide fragments of purified NMASP polypeptideare used. The peptides may be produced by protease digestion, chemicalcleavage of isolated or purified NMASP polypeptide or chemical synthesisand then may be isolated or purified. Such isolated or purified peptidescan be used directly as immunogens. In particular embodiments, usefulpeptide fragments are 5 or more amino acids in length and include, butare not limited to, those comprising the sequences LTNTHV (SEQ ID NO:5);SDVAL (SEQ ID NO:6) and GNSGGPL (SEQ ID NO:7).

Useful immunogens may also comprise such peptides or peptide fragmentsconjugated to a carrier molecule, preferably a carrier protein. Carrierproteins may be any commonly used in immunology, include, but are notlimited to, bovine serum albumin (BSA), chicken albumin, keyhole limpethemocyanin (KLH) and the like. For a discussion of hapten proteinconjugates, see, for example, Hartlow, et al., Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1988, or a standard immunology textbook such as Roitt, I. et al.,IMMUNOLOGY, C. V. Mosby Co., St. Louis, Mo. (1985) or Klein, J.,IMMUNOLOGY, Blackwell Scientific Publications, Inc., Cambridge, Mass.,(1990).

In yet another embodiment, for the production of antibodies thatspecifically bind one or more epitopes of the native NMASP polypeptide,intact Neisseria meningitidis cells or blebs prepared therefrom are usedas immunogen. The cells or blebs may be fixed with agents such asformaldehyde or glutaraldehyde before immunization. See Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988, Chapter 15. It is preferred that suchanti-whole cell antibodies be monoclonal antibodies. Hybridoma linesproducing the desired monoclonal antibodies can be identified by usingpurified NMASP polypeptide as the screening ligand. The immunogen forinducing these antibodies are whole cells, blebs, extracts or lysates ofany Neisseria meningitidis, including, but not limited to, types A-L andW. Preferred are N.m. Type A, Type B, Type C and Type W. Particularlypreferred are strains of N.m. Type A: ATCC13077, ATCC53417; Type BATCC13090, ATCC13091, ATCC13092, ATCC13093, ATCC13094, ATCC13096,ATCC13098, ATCC13100, ATCC23247, ATCC23249, ATCC23250, ATCC23251,ATCC23253, ATCC23254, ATCC23255, ATCC23583, ATCC33086, ATCC53044,ATCC53415, ATCC53418; Type C ATCC13102, ATCC13103, ATCC13105, ATCC13106,ATCC132107, ATCC13108, ATCC13109, ATCC13110, ATCC13111, ATCC13112,ATCC23252, ATCC23248, ATCC31275, ATCC53414, ATCC53416, ATCC53900; andType 29-E ATCC35558.

Polyclonal antibodies produced by whole cell or bleb immunizationscontain antibodies that bind other Neisseria meningitidis proteins(“non-anti-NMASP antibodies”) and thus are more cumbersome to use whereit is known or suspected that the sample contains other Neisseriameningitidis proteins or materials that are cross-reactive with theseother proteins. Under such circumstances, any binding by the anti-wholecell antibodies of a given sample or band must be verified bycoincidental binding of the same sample or band by antibodies thatspecifically bind NMASP polypeptide (e.g., anti-NMASP) and/or aNMASP-derived polypeptide, or by competition tests using anti-NMASPantibodies, NMASP polypeptide or NMASP-derived polypeptide as thecompetitor (i.e., addition of anti-NMASP antibodies, NMASP polypeptideor NMASP-derived polypeptide to the reaction mix lowers or abolishessample binding by anti-whole cell antibodies). Alternatively, suchpolyclonal antisera, containing “non-anti-NMASP” antibodies, may becleared of such antibodies by standard approaches and methods. Forexample, the non-anti-NMASP antibodies may be removed by precipitationwith cells of a NMASP deletion or “knock-out” mutant Neisseriameningitidis cultivars or Neisseria meningitidis strains known not tohave the NMASP polypeptide; or by absorption to columns comprising suchcells or outer membrane proteins of such cells.

In further embodiments, useful immunogens for eliciting antibodies ofthe invention comprise mixtures of two or more of any of theabove-mentioned individual immunogens.

Immunization of animals with the immunogens described herein, preferablyhumans, rabbits, rats, mice, sheep, goats, cows or horses, is performedfollowing procedures well known to those skilled in the art, forpurposes of obtaining antisera containing polyclonal antibodies orhybridoma lines secreting monoclonal antibodies.

Monoclonal antibodies can be prepared by standard techniques, given theteachings contained herein. Such techniques are disclosed, for example,in U.S. Pat. Nos. 4,271,145 and 4,196,265. Briefly, an animal isimmunized with the immunogen. Hybridomas are prepared by fusing spleencells from the immunized animal with myeloma cells. The fusion productsare screened for those producing antibodies that bind to the immunogen.The positive hybridomas clones are isolated, and the monoclonalantibodies are recovered from those clones.

Immunization regimens for production of both polyclonal and monoclonalantibodies are well known in the art. The immunogen may be injected byany of a number of routes, including subcutaneous, intravenous,intraperitoneal, intradermal, intramuscular, mucosal, or a combinationof these. The immunogen may be injected in soluble form, aggregate form,attached to a physical carrier, or mixed with an adjuvant, using methodsand materials well known in the art. The antisera and antibodies may bepurified using column chromatography methods well known to those ofskill in the art.

According to the present invention, NMASP polypeptides of Neisseriameningitidis strains are immuno-cross reactive. Thus, antibodies raisedto NMASP polypeptide of one Neisseria meningitidis species, strain orcultivar, specifically bind NMASP polypeptide and NMASP-derivedpolypeptides of other Neisseria meningitidis species, strains andcultivars. For example, polyclonal anti-NMASP antibodies induced byNMASP polypeptide of N.m. Type B specifically bind not only theidentical strain NMASP polypeptide (i.e., the NMASP polypeptide of N.m.Type B) but also NMASP polypeptide and/or NMASP-derived polypeptides ofother Neisseria meningitidis, including, but not limited to, types A andC-L and W. Preferred species are N.m. Type A, Type B, Type C and Type W.

The antibodies of the invention, including but not limited to anti-NMASPantibodies, can be used to facilitate isolation and purification ofNMASP polypeptide and NMASP-derived polypeptides. The antibodies mayalso be used as probes for identifying clones in expression librariesthat have inserts encoding NMASP polypeptide or fragments thereof. Theantibodies may also be used in immunoassays (e.g., ELISA, RIA, Westerns)to specifically detect and/or quantitate Neisseria meningitidis inbiological specimens. Thus anti-NMASP antibodies can be used to diagnoseNeisseria infections.

The antibodies of the invention, particularly those which are cytotoxic,may also be used in passive immunization to prevent or attenuateNeisseria meningitidis infections of animals, including humans. (As usedherein, a cytotoxic antibody is one which enhances opsonization and/orcomplement killing of the bacterium bound by the antibody). An effectiveconcentration of polyclonal or monoclonal antibodies raised against theimmunogens of the invention may be administered to a host to achievesuch effects. The exact concentration of the antibodies administeredwill vary according to each specific antibody preparation, but may bedetermined using standard techniques well known to those of ordinaryskill in the art. Administration of the antibodies may be accomplishedusing a variety of techniques, including, but not limited to thosedescribed in Section 5.6. for the delivery of vaccines.

Compositions

The present invention also provides therapeutic and prophylacticcompositions, which may be immunogenic compositions including vaccines,against Neisseria meningitidis infections of animals, including mammals,and more specifically rodents and primates, including humans. Preferredimmunogenic compositions include vaccines for use in humans. Theimmunogenic compositions of the present invention can be prepared bytechniques known to those skilled in the art and would comprise, forexample, an immunologically effective amount of any of the NMASPimmunogens disclosed in Section 5.4., optionally in combination with orfused to or conjugated to one or more other immunogens including lipids,phospholipids, lipopolysaccharides and other proteins of Neisserialorigin or other bacterial origin, a pharmaceutically acceptable carrier,optionally an appropriate adjuvant, and optionally other materialstraditionally found in vaccines. Such a cocktail vaccine (comprisingseveral immunogens) has the advantage that immunity against severalpathogens can be obtained by a single administration. Examples of otherimmunogens include, but are not limited to, those used in the known DPTvaccines, entire organisms or subunits therefrom of Neisseriameningitidis, Haemophilus influenzae, Moraxella catarrhalis, andStreptococcus pneumoniae, etc.

According to another embodiment, the immunogenic compositions of theinvention comprise an immunologically effective amount of one or more ofan inactivated or attenuated Neisseria meningitidis. An inactivated orattenuated Neisseria meningitidis is obtained using any methods known inthe art including, but not limited to, chemical treatment (e.g.,formalin), heat treatment and irradiation of Neisseria organisms.

The term “immunologically effective amount” is used herein to mean anamount sufficient to induce an immune response to produce antibodies, inthe case of a humoral immune response and/or cytokines and othercellular immune response components. Preferably, the immunogeniccomposition is one that prevents Neisseria meningitidis infections orattenuates the severity of any preexisting or subsequent Neisseriameningitidis infection. An immunologically effective amount of theimmunogen to be used in the vaccine is determined by means known in theart in view of the teachings herein. The exact concentration will dependupon the specific immunogen to be administered, but can be determined byusing standard techniques well known to those skilled in the art forassaying the development of an immune response.

Useful immunogens include the isolated NMASP polypeptide andNMASP-derived polypeptides of the present invention optionally incombination with or fused to or conjugated to one or more other antigensincluding lipids, phospholipids, lipopolysaccharides and other proteins.Preferred immunogens include the purified NMASP polypeptide andNMASP-derived polypeptides or peptides.

The combined immunogen and carrier or diluent may be an aqueoussolution, emulsion or suspension or may be a dried preparation. Ingeneral, the quantity of polypeptide immunogen will be between 0.1 and500 micrograms per dose. The carriers are known to those skilled in theart and include stabilizers, diluents, and buffers. Suitable stabilizersinclude carbohydrates, such as sorbitol, lactose, mannitol, starch,sucrose, dextran, and glucose and proteins, such as albumin or casein.Suitable diluents include saline, Hanks Balanced Salts, and Ringerssolution. Suitable buffers include an alkali metal phosphate, an alkalimetal carbonate, or an alkaline earth metal carbonate.

The immunogenic compositions, including vaccines, may also contain oneor more adjuvant or immunostimulatory compounds to improve or enhancethe immunological response. Suitable adjuvants include, but are notlimited to, peptides including bacterial toxins, such as but not limitedto heat labile toxin and/or verotoxin of E. coli, cholera toxin, andshiga toxin, and toxoids and/or attenuated forms thereof, chemokines,cytokines and the like; aluminum hydroxide; aluminum phosphate; aluminumoxide; a composition that consists of a mineral oil, such as Marcol 52,or a vegetable oil, and one or more emulsifying agents or surface activesubstances such as saponins, lysolecithin, polycations, polyanions; andpotentially useful human adjuvants such as BCG, QS21, MPL andCorynebacterium parvum.

The immunogenic compositions, including vaccines, of the invention areprepared by techniques known to those skilled in the art, given theteachings contained herein. Generally, an immunogen is mixed with thecarrier to form a solution, suspension, or emulsion. One or more of theadditives discussed above may be in the carrier or may be addedsubsequently. The vaccine preparations may be desiccated, for example,by freeze drying or spray drying for storage or formulations purposes.They may be subsequently reconstituted into liquid vaccines by theaddition of an appropriate liquid carrier or administered in dryformulation known to those skilled in the art, particularly in capsulesor tablet forms.

The immunogenic compositions, including vaccines, are administered tohumans or other animals, preferably other mammals, such as ruminants,rodents and primates. They can be administered in one or more doses. Thevaccines may be administered by known routes of administration. Manymethods may be used to introduce the vaccine formulations describedhere. These methods include but are not limited to oral, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, andintranasal routes. The preferred routes are intramuscular orsubcutaneous injection.

The invention also provides a method for inducing an immune response toNeisseria meningitidis in an animal to generate a humoral and/orcellular immune response. The method comprises administering animmunologically effective amount of an immunogen of the invention to ahost and, preferably, administering a vaccine of the invention to ahost.

Nucleic Acids Encoding the NMASP Polypeptide and NMASP-derivedPolypeptides

The present invention also provides nucleic acids, DNA and RNA, encodingNMASP polypeptide and NMASP-derived polypeptides and pharmaceuticalcompositions comprising same. In a particular embodiment, the NMASPpolypeptide comprises a deduced amino acid sequence as depicted in SEQID NOs: 2, 11 or 12 and the nucleic acids comprise nucleotide sequencesencoding said amino acid sequences. Fragments of NMASP have 5, 6, 7, 8,9 or more amino acids from those depicted in SEQ ID NOs: 2, 11 or 12 andthe nucleic acids comprise nucleotides encoding the same. Particularlypreferred fragments of NMASP have amino acid sequences depicted in SEQID NOs: 5-7, and 16 and the invention encompasses nucleic acidscomprising nucleotides encoding said amino acid sequences. In anotherparticular embodiment, the NMASP polypeptide is encoded by thenucleotide sequence of SEQ ID NOs: 1, 10 or 13, with particularlypreferred fragments depicted in SEQ ID NOs: 3, 4, 8, 9, 14, 15, and17-20.

Nucleic acids of the present invention can be single or double stranded.The invention also provides nucleic acids hybridizable to orcomplementary to the foregoing sequences. In specific aspects, nucleicacids are provided which comprise a sequence complementary to at least10, 25, 50, 100, 200, or 250 contiguous nucleotides of a nucleic acidencoding NMASP polypeptide or an NMASP-derived polypeptide. In aspecific embodiment, a nucleic acid which is hybridizable to a nucleicacid encoding NMASP polypeptide (e.g., having sequence SEQ. ID. NO.: 1,10 or 13), or to a nucleic acid encoding an NMASP-derived polypeptide,under conditions of low stringency is provided.

By way of example and not limitation, procedures using such conditionsof low stringency are as follows (see also Shilo and Weinberg, 1981,Proc. Natl. Acad. Sci. USA 78:6789-6792): Filters containing DNA arepretreated for 6 h at 40° C. in a solution containing 35% formamide,5×SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations arecarried out in the same solution with the following modifications: 0.02%PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol)dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 h at 40° C., and thenwashed for 1.5 h at 55° C. in a solution containing 2×SSC, 25 mMTris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution isreplaced with fresh solution and incubated an additional 1.5 h at 60° C.Filters are blotted dry and exposed for autoradiography. If necessary,filters are washed for a third time at 65-68° C. and re-exposed to film.Other conditions of low stringency which may be used are well known inthe art (e.g., as employed for cross-species hybridizations).

In another specific embodiment, a nucleic acid which is hybridizable toa nucleic acid encoding NMASP polypeptide or an NMASP-derivedpolypeptide under conditions of high stringency is provided. By way ofexample and not limitation, procedures using such conditions of highstringency are as follows: Prehybridization of filters containing DNA iscarried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC,50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA,and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48h at 65° C. in prehybridization mixture containing 100 μg/ml denaturedsalmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing offilters is done at 37° C. for 1 h in a solution containing 2×SSC, 0.01%PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by a wash in 0.1×SSCat 50° C. for 45 min before autoradiography. Other conditions of highstringency which may be used are well known in the art.

In another specific embodiment, a nucleic acid which is hybridizable toa nucleic acid encoding NMASP polypeptide or an NMASP-derivedpolypeptide under conditions of moderate stringency is provided.

Various other stringency conditions which promote nucleic acidhybridization can be used. For example, hybridization in 6×SSC at about45° C., followed by washing in 2×SSC at 50° C. may be used.Alternatively, the salt concentration in the wash step can range fromlow stringency of about 5×SSC at 50° C., to moderate stringency of about2×SSC at 50° C., to high stringency of about 0.2×SSC at 50° C. Inaddition, the temperature of the wash step can be increased from lowstringency conditions at room temperature, to moderately stringentconditions at about 42° C., to high stringency conditions at about 65°C. Other conditions include, but are not limited to, hybridizing at 68°C. in 0.5M NaHPO₄ (pH7.2)/1 mM EDTA/7% SDS, or hybridization in 50%formamide/0.25M NaHPO₄ (pH 7.2)/0.25 M NaCl/1 mM EDTA/7% SDS; followedby washing in 40 mM NaHPO₄ (pH 7.2)/1 mM EDTA/5% SDS at 42° C. or in 40mM NaHPO₄ (pH7.2) 1 mM EDTA/1% SDS at 50° C. Both temperature and saltmay be varied, or alternatively, one or the other variable may remainconstant while the other is changed.

Low, moderate and high stringency conditions are well known to those ofskill in the art, and will vary predictably depending on the basecomposition of the particular nucleic acid sequence and on the specificorganism from which the nucleic acid sequence is derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Second Edition, Cold SpringHarbor Press, New York, pp. 9.47-9.57; and Ausubel et al., 1989, CurrentProtocols in Molecular Biology, Green Publishing Associates and WileyInterscience, New York.

Nucleic acids encoding NMASP-derived polypeptides, including but notlimited to fragments or a portion thereof, (see Section 5.2), and NMASPantisense nucleic acids are additionally provided. As is readilyapparent, as used herein, a “nucleic acid encoding a fragment or portionof a nucleic acid encoding NMASP polypeptide or an NMASP-derivedpolypeptide” shall be construed as referring to a nucleic acid encodingonly the recited fragment or portion of the nucleic acid encoding NMASPpolypeptide or an NMASP-derived polypeptide and not the other contiguousportions of the nucleic acid encoding NMASP polypeptide or anNMASP-derived polypeptide protein as a continuous sequence.

Also encompassed are nucleotide sequences substantially homologous tothe above described nucleic acids. As used herein a “substantiallyhomologous” sequence is at least 70%, preferably greater than 80%, morepreferably greater than 90% identical to a reference sequence ofidentical size or when the alignment or comparison is conducted by acomputer homology program or search algorithm known in the art.

By way of example and not limitation, useful computer homology programsinclude the following: Basic Local Alignment Search Tool (BLAST)(Altschul et al., 1990, J. of Molec. Biol., 215:403-410, “The BLASTAlgorithm; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402) aheuristic search algorithm tailored to searching for sequence similaritywhich ascribes significance using the statistical methods of Karlin andAltschul (1990, Proc. Nat'l Acad. Sci. USA, 87:2264-68; 1993, Proc.Nat'l Acad. Sci. USA 90:5873-77). Five specific BLAST programs areprovided and the BLASTN program compares a nucleotide query sequenceagainst a nucleotide sequence database. Additional algorithms which canbe useful are the Smith-Waterman and FASTA algorithms. See supra Section5.1 for a more detailed description of useful algorithms and parametersfor determining percent identity of nucleotide (and/or amino acid)sequences.

In one aspect, the nucleic acids of the invention may be synthesizedusing methods known in the art. Specifically, a portion of or the entireamino acid sequence of NMASP polypeptide or an NMASP-derived polypeptidemay be determined using techniques well known to those of skill in theart, such as via the Edman degradation technique (see, e.g., Creighton,1983, Proteins: Structures and Molecular Principles, W. H. Freeman &Co., New York, pp.34-49). The amino acid sequence obtained is used as aguide for the synthesis of DNA encoding NMASP polypeptide orNMASP-derived polypeptide using conventional chemical approaches orpolymerase chain reaction (PCR) amplification of overlappingoligonucleotides.

In another aspect, the amino acid sequence may be used as a guide forsynthesis of oligonucleotide mixtures which in turn can be used toscreen for NMASP polypeptide coding sequences in Neisseria meningitidisgenomic libraries and PCR amplification products. Preferably the DNAused as the source of the NMASP polypeptide coding sequence, for bothgenomic libraries and PCR amplification, is prepared from cells of anyNeisseria meningitidis, including, but not limited to, types A-L and W.Preferred are N.m. Type A, Type B, Type C. and Type W. Strains from anyof these organisms may be obtained worldwide from any biologicalsdepository, particularly strains of N.m. Type A: ATCC13077, ATCC53417;Type B ATCC13090, ATCC13091, ATCC13092, ATCC13093, ATCC13094, ATCC13096,ATCC13098, ATCC13100, ATCC23247, ATCC23249, ATCC23250, ATCC23251,ATCC23253, ATCC23254, ATCC23255, ATCC23583, ATCC33086, ATCC53044,ATCC53415, ATCC53418; Type C ATCC13102, ATCC13103, ATCC13105, ATCC13106,ATCC132107, ATCC13108, ATCC13109, ATCC13110, ATCC13111, ATCC13112,ATCC23252, ATCC23248, ATCC31275, ATCC53414, ATCC53416, ATCC53900; andType 29-E ATCC35558.

In the preparation of genomic libraries, DNA fragments are generated,some of which will encode parts or the whole of Neisseria meningitidisNMASP polypeptide. The DNA may be cleaved at specific sites usingvarious restriction enzymes. Alternatively, one may use DNase in thepresence of manganese to fragment the DNA, or the DNA can be physicallysheared, as for example, by sonication and the like. The DNA fragmentscan then be separated according to size by standard techniques,including but not limited to, agarose and polyacrylamide gelelectrophoresis, column chromatography and sucrose gradientcentrifugation. The DNA fragments can then be inserted into suitablevectors, including but not limited to plasmids, cosmids, bacteriophageslambda or T₄, and yeast artificial chromosome (YAC). (See, for example,Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Glover,D. M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, U.K. Vol. I, II.) The genomic library may be screened by nucleicacid hybridization to labeled probe (Benton and Davis, 1977, Science196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A.72:3961).

The genomic libraries may be screened with a labeled degenerateoligonucleotide probe corresponding to the amino acid sequence of anypeptide fragment of the NMASP polypeptide using optimal approaches wellknown in the art. Any probe used preferably is 15 nucleotides or longer.Examples of particular probes are described below.

Clones in libraries with insert DNA encoding the NMASP polypeptide orfragments thereof will hybridize to one or more of the degenerateoligonucleotide probes. Hybridization of such oligonucleotide probes togenomic libraries are carried out using methods known in the art. Any ofthe hybridization procedures described in detail above in this Sectioncan be used. For a specific illustrative example, hybridization with thetwo above-mentioned oligonucleotide probes may be carried out in 2×SSC,1.0% SDS at 50_C and washed using the same conditions.

In yet another aspect, clones of nucleotide sequences encoding a part orthe entire NMASP polypeptide or NMASP-derived polypeptides may also beobtained by screening Neisseria meningitidis expression libraries. Forexample, Neisseria meningitidis DNA is isolated and random fragments areprepared and ligated into an expression vector (e.g., a bacteriophage,plasmid, phagemid or cosmid) such that the inserted sequence in thevector is capable of being expressed by the host cell into which thevector is then introduced. Various screening assays can then be used toselect for the expressed NMASP polypeptide or NMASP-derivedpolypeptides. In one embodiment, the various anti-NMASP antibodies ofthe invention (see Section 5.5) can be used to identify the desiredclones using methods known in the art. See, for example, Harlow andLane, 1988, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., Appendix IV. Clones orplaques from the library are brought into contact with the antibodies toidentify those clones that bind.

In an embodiment, colonies or plaques containing DNA that encodes NMASPpolypeptide or NMASP-derived polypeptide could be detected using DYNABeads according to Olsvick et al., 29th ICAAC, Houston, Tex. 1989,incorporated herein by reference. Anti-NMASP antibodies are crosslinkedto tosylated DYNA Beads M280, and these antibody-containing beads thenare used to adsorb to colonies or plaques expressing NMASP polypeptideor NMASP-derived polypeptide. Colonies or plaques expressing NMASPpolypeptide or NMASP-derived polypeptide is identified as any of thosethat bind the beads.

Alternatively, the anti-NMASP antibodies can be nonspecificallyimmobilized to a suitable support, such as protein A or G resins, silicaor Celite™ resin. This material is then used to adsorb to bacterialcolonies expressing NMASP polypeptide or NMASP-derived polypeptide asdescribed in the preceding paragraph.

In another aspect, PCR amplification may be used to producesubstantially pure DNA encoding a part of or the whole of NMASPpolypeptide from Neisseria meningitidis genomic DNA. Oligonucleotideprimers, degenerate or otherwise, corresponding to NMASP polypeptidesequences presently taught can be used as primers. In particularembodiments, a convergent set of oligonucleotides, degenerate orotherwise, specific for the NMASP coding sequences of SEQ ID NOs: 1, 10or 13 may be used to produce NMASP-encoding DNA.

As examples, an oligonucleotide encoding the N-terminal segment of theNMASP polypeptide and having the sequence 5′-GTG TTC AAA AAA TAC CAA TACCTC -3′ (SEQ ID NO: 18) may be used as the 5′ forward primer togetherwith a 3′ reverse PCR oligonucleotide complementary to an internal,downstream protein coding sequence having the sequence 5′-ACT GAC GCTGCC GTC GTC TTT GGT -3′ (SEQ ID NO: 19) may be used to amplify anN-terminal-specific NMASP DNA fragment. Alternatively, anoligonucleotide encoding an internal NMASP coding sequence and havingthe sequence: 5′-ATG CTG CTG CCC GAC TTT GTC CAA GTT CAA-3′ (SEQ ID NO:8) may be used as the 5′ forward PCR primer together with a 3′ reversePCR oligonucleotide complementary to downstream, internal NMASP proteincoding sequences and having the sequence 5′-GAA GCC CGA ACC GAA GTT CAATCC GCC GTC-3′ (SEQ ID NO: 9) may be used to PCR amplify an internalNMASP-specific DNA fragment. Alternatively forward primer SEQ ID NO: 20can be combined together with an oligonucleotide complementary to theC-terminal NMASP coding region and having the sequence 5′-TTG CAG GTTTAA TGC GAT AAA CAG CGT -3′ (SEQ ID NO: 20) to PCR amplify the NMASPORF. These NMASP-specific PCR products can be cloned into appropriateexpression vectors to direct the synthesis of all or part of the NMASPpolypeptide. Alternatively, these NMASP-specific PCR products can beappropriately labelled and used as hybridization probes to identify allor part of the NMASP gene from genomic DNA libraries.

PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermalcycler and Taq polymerase (Gene Amp™). One can choose to synthesizeseveral different degenerate primers, for use in the PCR reactions. Itis also possible to vary the stringency of hybridization conditions usedin priming the PCR reactions, to allow for greater or lesser degrees ofnucleotide sequence similarity between the degenerate primers and thecorresponding sequences in Neisseria meningitidis DNA. After successfulamplification of a segment of the sequence encoding NMASP polypeptide,that segment may be molecularly cloned and sequenced, and utilized as aprobe to isolate a complete genomic clone. This, in turn, permits thedetermination of the gene's complete nucleotide sequence, the analysisof its expression, and the production of its protein product forfunctional analysis, as described infra.

Once an NMASP polypeptide coding sequence has been isolated from oneNeisseria meningitidis species, strain or cultivar, it is possible touse the same approach to isolate NMASP polypeptide coding sequences fromother Neisseria meningitidis species, strains and cultivars. It will berecognized by those skilled in the art that the DNA or RNA sequenceencoding NMASP polypeptide (or fragments thereof) of the invention canbe used to obtain other DNA or RNA sequences that hybridize with itunder conditions of moderate to high stringency, using generaltechniques known in the art. Hybridization with an NMASP sequence fromone Neisseria meningitidis strain or cultivar under high stringencyconditions will identify the corresponding sequence from other strainsand cultivars. High stringency conditions vary with probe length andbase composition. The formulae for determining such conditions are wellknown in the art. See Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, N.Y., Chapter 11. As usedherein high stringency hybridization conditions as applied to probes ofgreater than 300 bases in length involve a final wash in 0.1×SSC/0.1%SDS at 68° C. for at least 1 hour (Ausubel, et al., Eds., 1989, CurrentProtocols in Molecular Biology, Vol. I, Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., New York, at page 2.10.3). Inparticular embodiments, the high stringency wash in hybridization usinga probe, for instance, having the sequence of SEQ ID NO:8 or 9 or itscomplement, is 2×SSC, 1% SDS at 50° C. for about 20 to about 30 minutes.

One skilled in the art would be able to identify complete clones ofNMASP polypeptide coding sequence using approaches well known in theart. The extent of NMASP polypeptide coding sequence contained in anisolated clone may be ascertained by sequencing the cloned insert andcomparing the deduced size of the polypeptide encoded by the openreading frames (ORFs) with that of NMASP polypeptide and/or by comparingthe deduced amino acid sequence with that of known amino acid sequenceof purified NMASP polypeptide. Where a partial clone of NMASPpolypeptide coding sequence has been isolated, complete clones may beisolated by using the insert of the partial clone as hybridizationprobe. Alternatively, a complete NMASP polypeptide coding sequence canbe reconstructed from overlapping partial clones by splicing theirinserts together.

Complete clones may be any that have ORFs with deduced amino acidsequence matching or substantially homologous to that of NMASPpolypeptide or, where the complete amino acid sequence of the latter isnot available, that of a peptide fragment of NMASP polypeptide andhaving a molecular weight corresponding to that of NMASP polypeptide.Further, complete clones may be identified by the ability of theirinserts, when placed in an expression vector, to produce a polypeptidethat binds antibodies specific to the amino-terminal of NMASPpolypeptide and antibodies specific to the carboxyl-terminal of NMASPpolypeptide.

Nucleic acid sequences encoding NMASP-derived polypeptides may beproduced by methods well known in the art. In one aspect, sequencesencoding NMASP-derived polypeptides can be derived from NMASPpolypeptide coding sequences by recombinant DNA methods in view of theteachings disclosed herein. For example, the coding sequence of NMASPpolypeptide may be altered creating amino acid substitutions that willnot affect the immunogenicity of the NMASP polypeptide or which mayimprove its immunogenicity, such as conservative or semi-conservativesubstitutions as described above. Various methods may be used, includingbut not limited to oligonucleotide directed, site specific mutagenesis.These and other techniques known in the art may be used to create singleor multiple mutations, such as replacements, insertions, deletions, andtranspositions, as described in Botstein and Shortle, 1985, Science229:1193-1210.

Further, DNA of NMASP polypeptide coding sequences may be truncated byrestriction enzyme or exonuclease digestions. Heterologous codingsequence may be added to NMASP polypeptide coding sequence by ligationor PCR amplification. Moreover, DNA encoding the whole or a part of anNMASP-derived polypeptide may be synthesized chemically or using PCRamplification based on the known or deduced amino acid sequence of NMASPpolypeptide and any desired alterations to that sequence.

The identified and isolated DNA containing NMASP polypeptide orNMASP-derived polypeptide coding sequence can be inserted into anappropriate cloning vector. A large number of vector-host systems knownin the art may be used. Possible vectors include, but are not limitedto, plasmids and modified viruses, but the vector system must becompatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas pTrcHis, pBR322 or pUC plasmid derivatives. The insertion into acloning vector can, for example, be accomplished by ligating the DNAfragment into a cloning vector which has complementary cohesive termini.However, if the complementary restriction sites used to fragment the DNAare not present in the cloning vector, the ends of the DNA molecules maybe enzymatically modified. Alternatively, any site desired may beproduced by ligating nucleotide sequences (linkers) onto the DNAtermini; these ligated linkers may comprise specific chemicallysynthesized oligonucleotides encoding restriction endonucleaserecognition sequences. In an alternative method, the cleaved DNA may bemodified by homopolymeric tailing. Recombinant molecules can beintroduced into host cells via transformation, transfection, infection,electroporation, etc., so that many copies of the gene sequence aregenerated.

In an alternative method, the desired DNA containing NMASP polypeptideor NMASP-derived polypeptide coding sequence may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired sequence, for example, by sizefractionation, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that contain NMASP polypeptide or NMASP-derivedpolypeptide coding sequence enables generation of multiple copies ofsuch coding sequence. Thus, the coding sequence may be obtained in largequantities by growing transformants, isolating the recombinant DNAmolecules from the transformants and, when necessary, retrieving theinserted coding sequence from the isolated recombinant DNA.

Recombinant Production of NMASP Polypeptide and NMASP-derivedPolypeptides

NMASP polypeptide and NMASP-derived polypeptides of the invention may beproduced through genetic engineering techniques. In this case, they areproduced by an appropriate host cell that has been transformed by DNAthat codes for the polypeptide. The nucleotide sequence encoding NMASPpolypeptide or NMASP-derived polypeptides of the invention can beinserted into an appropriate expression vector, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted polypeptide-coding sequence. The nucleotide sequencesencoding NMASP polypeptide or NMASP-derived polypeptides are insertedinto the vectors in a manner that they will be expressed underappropriate conditions (e.g., in proper orientation and correct readingframe and with appropriate expression sequences, including an RNApolymerase binding sequence and a ribosomal binding sequence).

A variety of host-vector systems may be utilized to express thepolypeptide-coding sequence. These include but are not limited tomammalian cell systems infected with virus (e.g., vaccinia virus,adenovirus, etc.); insect cell systems infected with virus (e.g.,baculovirus); microorganisms such as yeast containing yeast vectors, orbacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA.Preferably, the host cell is a bacterium, and most preferably thebacterium is E. coli, B. subtilis or Salmonella.

The expression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system utilized, any one ofa number of suitable transcription and translation elements may be used.In a specific embodiment, a chimeric protein comprising NMASPpolypeptide or NMASP-derived polypeptide sequence and a pre and/or prosequence of the host cell is expressed. In other specific embodiments, achimeric protein comprising NMASP polypeptide or NMASP-derivedpolypeptide sequence and an affinity purification peptide is expressed.In further specific embodiments, a chimeric protein comprising NMASPpolypeptide or NMASP-derived polypeptide sequence and a usefulimmunogenic peptide or polypeptide is expressed. In preferredembodiments, NMASP-derived polypeptide expressed contains a sequenceforming either an outer-surface epitope or the receptor-binding domainof native NMASP polypeptide.

Any method known in the art for inserting DNA fragments into a vectormay be used to construct expression vectors containing a chimeric geneconsisting of appropriate transcriptional/translational control signalsand the polypeptide coding sequences. These methods may include in vitrorecombinant DNA and synthetic techniques and in vivo recombinants(genetic recombination). Expression of a nucleic acid sequence encodingNMASP polypeptide or NMASP-derived polypeptide may be regulated by asecond nucleic acid sequence so that the inserted sequence is expressedin a host transformed with the recombinant DNA molecule. For example,expression of the inserted sequence may be controlled by anypromoter/enhancer element known in the art. Promoters which may be usedto control expression of inserted sequences include, but are not limitedto the SV40 early promoter region (Bemoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42) for expression in animalcells; the promoters of lactamase (Villa-Kamaroff et al., 1978, Proc.Natl. Acad. Sci. U.S.A. 75:3727-3731), tac (DeBoer et al., 1983, Proc.Natl. Acad. Sci. U.S.A. 80:21-25), _P_(L), or trc for expression inbacterial cells (see also “Useful proteins from recombinant bacteria” inScientific American, 1980, 242:74-94); the nopaline synthetase promoterregion or the cauliflower mosaic virus 35S RNA promoter (Gardner et al.,1981, Nucl. Acids Res. 9:2871), and the promoter of the photosyntheticenzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984,Nature 310:115-120) for expression implant cells; promoter elements fromyeast or other fungi such as the Gal4 promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter.

Expression vectors containing NMASP polypeptide or NMASP-derivedpolypeptide coding sequences can be identified by three generalapproaches: (a) nucleic acid hybridization, (b) presence or absence of“marker” gene functions, and (c) expression of inserted sequences. Inthe first approach, the presence of a foreign gene inserted in anexpression vector can be detected by nucleic acid hybridization usingprobes comprising sequences that are homologous to the inserted NMASPpolypeptide or NMASP-derived polypeptide coding sequence. In the secondapproach, the recombinant vector/host system can be identified andselected based upon the presence or absence of certain “marker” genefunctions (e.g., thymidine kinase activity, resistance to antibiotics,transformation phenotype, occlusion body formation in baculovirus, etc.)caused by the insertion of foreign genes in the vector. For example, ifthe NMASP polypeptide or NMASP-derived polypeptide coding sequence isinserted within the marker gene sequence of the vector, recombinantscontaining the insert can be identified by the absence of the markergene function. In the third approach, recombinant expression vectors canbe identified by assaying the foreign gene product expressed by therecombinant. Such assays can be based, for example, on the physical orfunctional properties of NMASP polypeptide or NMASP-derived polypeptidein in vitro assay systems, e.g., binding to an NMASP ligand or receptor,or binding with anti-NMASP antibodies of the invention, or serineprotease activity.

Once a particular recombinant DNA molecule is identified and isolated,several methods known in the art may be used to propagate it. Once asuitable host system and growth conditions are established, recombinantexpression vectors can be propagated and prepared in quantity. Asexplained above, the expression vectors which can be used include, butare not limited to, the following vectors or their derivatives: human oranimal viruses such as vaccinia virus or adenovirus; insect viruses suchas baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), andplasmid and cosmid DNA vectors, to name but a few.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered NMASP polypeptide orNMASP-derived polypeptide may be controlled. Furthermore, different hostcells have characteristic and specific mechanisms for the translationaland post-translational processing and modification of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed.

Applications

The present invention has many utilities. For example, the NMASPpolypeptide and NMASP-derived polypeptides may be used as ligands todetect antibodies elicited in response to Neisseria meningitidisinfections (e.g., as a diagnostic marker in diagnosing Neisseriameningitidis infections). The NMASP polypeptide and NMASP-derivedpolypeptides may also be used as immunogens for inducing Neisseriameningitidis-specific antibodies. Such antibodies are useful inimmunoassays to detect Neisseria meningitidis in biological specimens.The cytotoxic antibodies of the invention are useful in passiveimmunizations against Neisseria meningitidis infections. The NMASPpolypeptide, NMASP-derived polypeptides, and/or fragments thereof mayfurther be used as active ingredients in vaccines against Neisseriameningitidis infections.

Not intending to be limited to any particular mechanism of action, theinventors provide the following remarks. The interaction of both normaland neoplastic mammalian cells with extracellular matrix components(ECM) such as fibronectin, vitronectin, and type I collagen has beenshown to be mediated through a family of cell-surface receptors thatspecifically recognize an arginine-glycine-aspartic acid amino acidsequence within each protein (Ruoslahti E. and M. D. Pierschbacher.1986. Arg-Gly-Asp: a versatile cell recognition signal. Cell 44:517-8).Numerous studies have shown that synthetic peptides containing theArg-Gly-Asp sequence can inhibit these receptor-ligand interactions invitro (Gehlsen K. R. et al. 1988. Inhibition of in vitro tumor cellinvasion by Arg-Gly-Asp-containing synthetic peptides. J. Cell Biol.106:925-30). A highly active Arg-Gly-Asp sequence has been identifiedwithin the cell attachment region of fibronectin and the interactionbetween this sequence and specific platelet cell surface receptors hasbeen demonstrated to induce activation. The conserved Arg-Gly-Asp andArg-Gly-Asn motifs reside near the C-terminus of the NMASP polypeptideof the present invention may also function as adherence domains specificfor ECM proteins. If so, once the NMASP polypeptide of the presentinvention is bound to the host's cellular matrix the proteolyticactivity of NMASP could function to remodel the epithelial/endothelialsurface so as to promote attachment and or subsequent invasion. Thususing the NMASP polypeptides of the invention as a vaccine to produceantibody that could interrupt these processes would be beneficial.

The polypeptides, peptides, antibodies, nucleic acids and vectorscomprising the nucleic acids, of the invention are useful as reagentsfor clinical or medical diagnosis of Neisseria meningitidis infectionsand for scientific research on the properties of pathogenicity,virulence, and infectivity of Neisseria meningitidis, as well as hostdefense mechanisms. For example, DNA and RNA of the invention can beused as probes to identify the presence of Neisseria meningitidis inbiological specimens by hybridization or PCR amplification. The DNA andRNA can also be used to identify other bacteria that might encode apolypeptide related to the Neisseria meningitidis NMASP.

The polypeptides and peptides of the invention may be used to preparepolyclonal and monoclonal antibodies that can be used to further purifycompositions containing the polypeptides of the invention by affinitychromatography. The polypeptides and peptides can also be used instandard immunoassays as diagnostics to screen for the presence ofantibodies to Neisseria meningitidis in a sample.

The nucleic acids, polypeptides and peptides of the invention are alsouseful in screening assays to detect compounds, including smallmolecules, or agents that are useful as diagnostic, therapeutic orprophylactic agents against Neisseria meningitidis infection. In oneillustrative mode of this embodiment, assays can be used to screen for amolecule or agent that binds to NMASP and hence which is useful as adiagnostic agent to detect Neisseria meningitidis in a patient bodilyfluid or tissue sample. In another illustrative mode of this embodiment,assays can be used to screen for a molecule or agent that targets NMASPpolypeptide or the nucleic acid encoding NMASP polypeptide and hencewhich molecule or agent is useful as an antibacterial agent for therapyor prophylaxis against Neisseria meningitidis infection. While notintending to be limited to any particular mode of action for theantibacterial agents identified according to the present invention, theinventors provide the following remarks. The novel NMASP polypeptide ofthe present invention has some limited sequence similarity to E. coliHtrA or DegP, including, but not limited to, conserved Arg-Gly-Asp andArg-Gly-Asn motifs near the C-terminus of the NMASP polypeptide. Theinventors envisage that molecules or agents that bind to, interact with,or inhibit the synthesis or enzymatic activity, such as but not limitedto, serine protease activity, of the NMASP polypeptide of the inventionare useful as anti-infective agents against Neisseria meningitidisinfection. Any assays known to those skilled in the art can be usedaccording to this embodiment to screen for such agents. Non-limitingillustrative examples of assays include the following.

A number of systems have been described which can be adapted for theidentification of agents interacting with NMASP polypeptide or NMASPderived polypeptides. One well known system is the yeast two-hybridsystem (Fields and Song, 1989, Nature 340:245-246; White. 1996, Proc.Natl. Acad. Sci. USA 93:10001-10003; Warbick, 1997, Structure 5:13-17)which has been used to identify interacting proteins and to isolate thecorresponding encoding genes. In this system, prototrophic selectablemarkers which allow positive growth selection are used as reporter genesto facilitate identification of protein-protein interactions. Applyingthe above general scheme, growing yeast cell colonies expressingDB-X/AD-Y-interacting proteins can be identified among the non-growingcolonies (Gyris et al., 1993, Cell 75:791-803; Durfee et al., 1993,Genes Dev. 7:555-569; Vojtek et al., 1993, Cell 74:205-214). Relatedsystems which may be employed include the yeast three-hybrid system(Licitra and Liu, 1996, Proc. Natl. Acad. Sci. USA 93:12817-12821;Tirode et al., 1997, J. Biol. Chem. 272:22995-22999) and the yeastreverse two-hybrid system (Vidal et al., 1996, Proc. Natl. Acad. Sci.USA 93:10321-10326; Vidal et al., 1996, Proc. Natl. Acad. Sci. USA93:10315-10320).

Bacterial systems for identification of protein-protein interactions arealso known in the art and may be adapted for use with the methods of thepresent invention. For example, in one embodiment, the E. coliCadC-based dimer detection system may be used for identifying proteinsinteracting with NMASP (see generally, PCT publication no. WO 99/23116dated May 14, 1999, which is incorporated herein in its entirety). Inanother embodiment, a bacterial protein interaction system based on theAraC protein, which regulates the L-arabinose operon in E. coli, may beused (Bustos and Schleif, 1993, Proc. Natl. Acad. Sci. USA 90:5638-5642;Soisson et al., 1997, Science 276:421-425; Eustance et al., 1994, J.Mol. Biol. 242:330-338). Other assay systems which may be used includebacterial systems based on the lambda repressor system (Zeng et al.,1997, Protein Sci. 6:2218-2226), the lac-operon (Gates et al., 1996, J.Mol. Biol. 255:373-386), an interaction signal detection based on lambdaand lambdoid proteins (Hollis et al., 1988. Proc. Natl. Acad. Sci. USA85:5834-5838), systems based on E. coli RNAP (Dove et al., 1998, GenesDev. 12:745-754; Dove et al., 1997, Nature 386:627-630), and systemsbased on the cAMP synthetase (Karimova et al., 1998, Proc. Natl. Acad.Sci. USA 95:5752-5756).

Alternatively, assays screening for interaction of molecules with NMASPcan be devised using a detectible marker. Proteins or other moleculesmay be labeled with a detectable marker using methods for proteinlabeling known in the art. A “detectable marker” refers to a moiety,such as a radioactive isotope or group containing same, or nonisotopiclabels, such as enzymes, biotin, avidin, streptavidin, digoxygenin,luminescent agents, dyes, haptens, and the like. Luminescent agents,depending upon the source of exciting energy, can be classified asradioluminescent, chemiluminescent, bioluminescent, and photoluminescent(including fluorescent and phosphorescent). An affinity capture assaymay be used.

In another embodiment, any molecule including macromolecules and smallmolecules, can be assayed for interaction with NMASP polypeptide or anNMASP-derived polypeptide; interaction with NMASP or an NMASP-derivedpolypeptide indicates the molecule is useful as a diagnostic,therapeutic or prophylactic against Neisseria meningitidis infection. Inone embodiment, the method is as follows. A method for assaying for anagent that interacts with NMASP polypeptide comprises: (a) contacting acell expressing NMASP polypeptide with an agent labeled with adetectable marker for a time sufficient to allow the agent to interactwith the polypeptide; (b) washing the cells; and (c) detecting anymarker associated with the cells, in which any cell associated markerindicates that the agent interacts with the NMASP polypeptide andwherein any agent that interacts with NMASP indicates that the agent isuseful as a diagnostic, prophylactic or therapeutic agent againstNeisseria meningitidis infection.

DNA or polypeptides of the invention may be used to assess the bindingof small molecule substrates and ligands in, for example, cells, cellfree preparations, chemical libraries, and natural product extracts andmixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics thereof.

The invention also provides a method of screening compounds to identifythose which enhance (i.e., agonists) or block (i.e., antagonists) theaction of NMASP polypeptides, particularly those compounds that arebacteriostatic or bactericidal to Neisseria meningitidis. The method ofscreening may involve high-throughput assay techniques. For example, toscreen for agonists or antagonists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any mixture thereof, comprising NMASP polypeptide and alabeled substrate or ligand such polypeptide is incubated in the absenceor the presence of a candidate molecule that may be a NMASP agonist orantagonist. The ability of the candidate molecule to agonize orantagonize the NMASP polypeptide is reflected in decreased binding ofthe labeled ligand or decreased production of product from suchsubstrate. Molecules that bind gratuitously, i.e., without inducing theeffects of NMASP polypeptide are most likely to be good antagonists.Molecules that bind well and increase the rate of product productionfrom substrate are agonists. Detection of the rate or level ofproduction of product from substrate may be enhanced by using a reportersystem. Reporter systems that may be useful in this regard include butare not limited to colorimetric labeled substrate converted intoproduct, a reporter gene that is responsive to change an NMASPpolypeptide activity, and binding assays known in the art. Potentialantagonists or agonists include small molecules, peptides, andantibodies that bind to a NMASP peptide or polypeptide of the invention,or such a closely related protein or antibody that binds the same siteson a binding molecule.

It is to be understood that the application of the teachings of thepresent invention to a specific problem or environment will be withinthe capabilities of one having ordinary skill in the art in light of theteachings contained herein.

5.9 The Above Disclosure Generally Describes the Present Invention

A more specific description of certain embodiments is provided below inthe following examples. The examples are described solely for thepurpose of illustration and are not intended to limit the scope of theinvention. Changes in form and substitution of equivalents arecontemplated as circumstances suggest or render expedient. Althoughspecific terms have been employed herein, such terms are intended in adescriptive sense and not for purposes of limitation.

Methods of molecular genetics, protein biochemistry and immunology usedbut not explicitly described in the disclosure and examples are amplyreported in the scientific literature and are well within the ability ofthose skilled in the art.

EXAMPLE: ISOLATION AND CHARACTERIZATION OF THE NMASP POLYPEPTIDE ANDNUCLEIC ACID ENCODING SAME Extraction of Envelope Proteins

Neisseria meningitidis are grown at 37° C. at 200 rpm in 1 liter ofMueller Hinton broth, chocolate agar plates or Columbia blood agarplates. Extraction with hypotonic solutions is carried out as follows.Cells are harvested into lithium chloride (LiCl), sodium acetate (NaOAc)solution (0.1M LiCl, 0.2 M NaOAc, pH 5.8) and shaken with glass beadsfor 3 h in a 45° C. water bath. The beads and cellular debris areremoved by centrifugation and for crude extracts, the supernatantremoved and stored at −20° C. For purified extracts, the supernatant isfurther centrifuged at 100,000×g and the resulting pellet resuspendedand either stored or used for further purification as described herein.

Extraction using detergents is carried out as follows. Cells areharvested into a Tris-hydrochloride buffer solution and pelleted bycentrifugation. The pelleted cells are resuspended and sonicated todisrupt the cells. Unbroken cells are removed by low speedcentrifugation and the total cell envelope fraction is treated witheither (1.25% final w/v) n-octyl-D-glucopyranoside (i.e., octylglucoside; OG) in phosphate buffered saline (PBS) or (0.5% w/v) ofsodium N-lauroyl sarcosine (Sarkosyl) for 30 minutes at roomtemperature. The unsolublized fraction is pelleted and the supernatantis used as the detergent extract for resolution using SDS-PAGE or forfurther purification as described herein.

Amino Terminal Sequencing of NMASP Polypeptide

NMASP polypeptide from extracts of Neisseria meningitidis is detected(e.g., by silver staining or anti-NMASP antibodies) in denaturing gels.For N-terminal sequencing, an extract is mixed with PAGE sample buffercontaining SDS, and is incubated for 3 minutes in boiling water bath.The proteins are then resolved on a PAG with SDS and transferred to aPVDF membrane by electroblotting. The region of the membrane containingthe NMASP band is then cut out and amino-terminal sequencing isperformed by generally accepted methods known to those skilled in theart.

Anti-NMASP Antiserum

Antisera to NMASP are prepared by injecting the NMASP polypeptide intoan animal, such as a rabbit, mouse or guinea pig, with or without anadjuvant. For instance, NMASP is injected with Freund's completeadjuvant followed by injections of NMASP with Freund's incompleteadjuvant. Normally, a semi-purified or purified form of the protein isinjected. For instance, the NMASP polypeptide is resolved from otherproteins using a denaturing sodium dodecylsulfate polyacrylamide gelaccording to standard techniques well known to those skilled in the art,as previously described (Laemmli, 1970, Nature 227:680-685), and cuttingthe NMASP-containing band out of the gel. The excised band containingNMASP is macerated and injected into an animal to generate antiserum tothe NMASP polypeptide. The antisera is examined using well known andgenerally accepted methods of ELISA to determine titres, by westernblots to determine binding to proteins, for immunofluorescent stainingand for complement-mediated cytotoxic activity against Neisseria asdescribed below.

Western Blots

N. meningitidis ATCC 13090 are grown on gonococcal agar (GC/agar base,Difco; supplemental with 1% Iso Vitale X, BBL) or chocolate agar platesfor 24-48 hours at 37° C. in 5% CO₂. Cells are removed by scraping thecolonies from the agar surface using a polystyrene inoculating loop.Cells are then solubilized by suspending 30 μg of cells in 150 μl ofPAGE sample buffer (360 mM Tris buffer [pH 8.8], containing2-mercaptoethanol, 4% sodium dodecylsulfate and 20% glycerol), andincubating the suspension at 100° C. for 5 minutes. The solubilizedcells are resolved on 12% polyacrylamide gels as per Laemmli and theseparated proteins were electrophoretically transferred to PVDFmembranes at 100 V for 1.5 hours as previously described (Thebaine etal. 1979, Proc. Natl. Acad. Sci. USA 76:4350-4354). The PVDF membranesare then pretreated with 25 ml of Dulbecco's phosphate buffered salinecontaining 0.5% sodium casein, 0.5% bovine serum albumin and 1% goatserum. All subsequent incubations are carried out using thispretreatment buffer.

PVDF membranes are incubated with 25 ml of a 1:500 dilution of preimmunerabbit serum or serum from a rabbit immunized with NMASP or Hin47polypeptide (as described above) for 1 hour at room temperature ormonoclonal antibodies to NMASP or to Hin47 (described above). PVDFmembranes are then washed twice with wash buffer (20 mM Tris buffer (pH7.5.) containing 150 mM sodium chloride and 0.05% TWEEN-20™(polyoxethenlenesorbitan monolaueate. PVDF membranes are incubated with25 ml of a 1:5000 dilution of peroxidase-labeled goat anti-rabbit (oranti-mouse for monoclonals) IgG (Jackson ImmunoResearch Laboratories,West Grove, Pa.) for 30 minutes at room temperature. PVDF membranes arethen washed 4 times with wash buffer, and are developed with3,3′diaminobenzidine tetrahydrochloride and urea peroxide as supplied bySigma Chemical Co. (St. Louis, Mo. catalog number D-4418) for 4 minuteseach.

Anti-NMASP Immunofluorescence Staining of Cell Surface

Neisseria meningitidis are grown overnight at 37° C. in a shaking waterbath in Mueller Hinton broth or on gonococcal agar and harvested byscraping. The cells are pelleted by centrifugation and then resuspendedin an equal volume of Dulbecco's modification of phosphate bufferedsaline without calcium or magnesium (PBS/MC). 20 μl of the cellsuspension is applied to each of 5 clean microscope slides. Aftersetting for 10 seconds, the excess fluid is removed with amicropipettor, and the slides are allowed to air dry for 1 hour. Theslides are then heat fixed over an open flame until the glass is warm tothe touch. The slides are initially treated with 40 μl of 1:40 dilutionof anti-NMASP antiserum or preimmune serum from the same animal dilutedin PBS/MC, or PBS/MC for 10 minutes, then washed 5 times with PBS/MC.The slides are treated with 40 μl of 5 μg/ml PBS/MC of fluoresceinisothiocyanate-labeled goat antibody to rabbit IgG (Kirkegaard and PerryLaboratories, Inc, Gaithersburg, Md.). The slides are incubated in thedark for 10 minutes and are washed 5 times in PBS/MC. Each slide isstored covered with PBS/MC under a cover slide and is viewed with afluorescence microscope fitted with a 489 nm filter. For each samplefive fields-of-view are visually examined to evaluate the extent ofstraining.

Cellular Envelope Location of NMASP

Rabbit anti-NMASP antiserum is used in indirect immunofluorescencestaining to determine if NMASP polypeptide is exposed on the outersurface of Neisseria meningitidis cells. This would indicate that inintact Neisseria meningitidis cells NMASP polypeptide is reactive withanti-NMASP antibodies.

Properties of NMASP Polypeptide

NMASP polypeptide exists as a protein of approximately 40-55 kD in itsnative state as can be determined using detergent or hypotonic extractsof Neisseria meningitidis, incubating the extracts with sodium dodecylsulfate at 100° C., and resolving the proteins on a denaturingpolyacrylamide gel.

Western blot analysis of protein extracts of a number of Neisseriameningitidis strains can be used to show that the anti-NMASP antibodiesbind to a polypeptide of about 40 kD to about 55 kD in many Neisseriameningitidis strains. Anti-NMASP antibodies may be used to specificallyidentify Neisseria meningitidis. NMASP polypeptide may be used togenerate antibodies that have diagnostic application for identificationof Neisseria meningitidis. Antibodies to NMASP polypeptide of onespecies or strain may be used to identify and isolate the correspondingNMASP polypeptide of other Neisseria meningitidis species or strains.

Example: EFFICACY OF NMASP VACCINE: CYTOTOXIC ACTIVITY OF ANTI-NMASPANTISERUM

Complement-mediated cytotoxic activity of anti-NMASP antibodies isexamined to determine the vaccine potential of NMASP polypeptide.Antiserum to NMASP polypeptide is prepared as described in Section6.1.8. supra. The activities of the pre-immune serum and the anti-NMASPantiserum in mediating complement killing of Neisseria meningitidis areexamined using the “Serum Bactericidal Test” described by Zollinger etal. (Immune Responses to Neisseria meningitis, in Manual of ClinicalLaboratory Immunology, 3rd ed., pg 347-349).

The results could be used to show that anti-NMASP antiserum mediatescomplement-killing of Neisseria meningitidis.

EXAMPLE: ISOLATION OF THE NMASP NUCLEIC ACID Identification of an NMASPOpen Reading Frame

The E. coli DegP (HtrA) amino acid sequence available from GeneBank wasemployed as a BLAST (TBLASTN) subject query to search the partiallycompleted, crude, and unassembled publicly available genomic sequencedatabases for N. meningitidis sero-group A (Sanger Center, UK) toidentify linear amino acid sequences that might share some similarity tothe DegP protein. No predicted amino acid sequences from these Neisseriadatabases showed more than ˜36% sequence identity to the E. coli DegPprotein sequence. [% identity determined using TBLASTN program (Altschulet al., 1990, J. Molec. Biol. 215:403-10; Altschul et al., 1997, Nuc.Acids Res. 25:3389-3402) with data entered using FASTA format; expect 10filter default; description 100, alignment]. Candidate NMASP amino acidsequences from the N. meningitidis A database were localized withinspecific genomic DNA sequence “contigs”, and putative open readingframes encoding these NMASP sequences were derived. Putative ORFscapable of encoding proteins of ˜40-55 kD, the average size of mostDegP-like serine proteases, were then selected and further analyzed forthe presence and appropriate relative spacing of semi-conservedcatalytic residues (H, D, S) thought to be required for serine proteaseactivity. A single putative open reading frame from the N. meningitidisA database was identified which met these criteria. This putative NMASPORFs were then compared to each other using a CLUSTAL pairwise analysisand found to be ˜96% identical at the primary amino acid level. Theseputative ORFs were then used to individually search the partiallycompleted N. meningitidis B genomic database (TIGR, USA) for similarputative NMASP amino acid sequences using the TBLASTN algorithm. Theseanalyses demonstrated that N. meningitidis B strain, like the N.meningitidis A, also contains a putative NMASP ORF that is highlyconserved (˜97%) compared to those identified in N. meningitidis A.

Isolation of N. Meningitidis Chromosomal DNA

N. meningitidis was streaked on gonococcal agar base (GC agar, Difco)containing 1.0% IsoVitale X (BBL) and grown at 35-37° C. in 5% CO₂ for˜24-48 hours. To prepare confluent “lawns” of cells for DNA isolation,three or four single colonies were picked from the “overnight” seedplate and used to inoculate fresh GC plates which were again grownovernight at 35-37° C. in 5% CO₂. Cells were collected from the surfaceof the agar plates by gentle rinsing using trypticase soy broth (TSB)containing 10% glycerol and then stored at −20° C. When needed, cellswere thawed at room temperature and bacteria collected by centrifugationin a Sorval SS34 rotor at ˜2000×g for 15 minutes at room temperature.The supernatant was removed and the cell pellet suspended in ˜5.0 ml ofsterile water. An equal volume of lysis buffer (200 mM NaCl, 20 mM EDTA,40 mM Tris-HCl pH 8.0, 0.5% (w/v) SDS, 0.5% (v/v) 2-mercaptoethanol, and250 ug/ml of proteinase K) was added and the cells suspended by gentleagitation and trituration. The cell suspension was then incubated ˜12hours at 50° C. to lyse the bacteria and liberate chromosomal DNA.Proteinaceous material was precipitated by the addition of 5.0 ml ofsaturated NaCl (˜6.0 M, in sterile water) and centrifugation at ˜5,500×gin a Sorval SS34 rotor at room temperature. Chromosomal DNA wasprecipitated from the cleared supernatant by the addition of two volumesof 100% ethanol. Aggregated DNA was collected and washed using gentleagitation in a small volume of a 70% ethanol solution. Purifiedchromosomal DNA was suspended in sterile water and allowed todissolve/disburse overnight at 4° C. by gentle rocking. Theconcentration of dissolved DNA was determined spectrophotometrically at260 nm using an extinction coefficient of 1.0 O.D. unit ˜50 μg/ml.

PCR CLONING OF THE NMASP ORF

Oligonucleotide PCR primers complementary to the DNA sequences encodingthe — and C-termini of the N. meningitidis A NMASP ORF present in theSanger database were synthesized. In addition to the NMASP specificsequences, these PCR primers were designed to contain flanking NcoI andEcoRI restriction sites in an effort to expedite cloning of the ORF.These oligonucleotides were used to amplify NMASP-specific PCR productsfrom three different, clinically relevant N. meningitidis B strains;H44/76, M96-250338, and BZ198.

The forward primer used for these PCR reactions was designatedNMASP-1-Nco (49 mer, forward primer) and NMASP-1-RI (54 mer, reverseprimer) and contain sequences complementary to 9 N-terminal and last 9C-terminal residues, respectively, of the putative NMASP protein. Inaddition to the NMASP coding sequences, the forward primer was designedto contain a unique NcoI restriction site optimally located upstream ofthe first Met residue of the NMASP protein while the reverse primer wasdesigned to contain an EcoRI restriction site immediately downstream ofthe TAA termination codon. These restriction sites were engineered toallow directional cloning and subsequent expression of the NMASP ORFfrom the commercially available procaryotic expression vector pTrcHis(InvitroGen). In order to introduce a correctly positioned NcoI site(CCATGG) at the N-terminus of the ORF, it was necessary to change thefirst base of the second codon (CTC) from C to G which effects aconservative residue substitution at this position (Leu=>Val).

NMASP-1-Nco (SEQ ID No.3) 5′-AAG GGC CCA ATT ACG CAG AGC CAT GGT GCT GCCCGA CTT TGT CCA ACT G-3′ NMASP-1-RI (SEQ ID No.4) 5′-AAG GGC CCA ATT ACGCAG AGG GAA TTC TTA TTG CAG GTT TAA TGC GAT AAA CAG-3′

Standard PCR amplification reactions (2 mM Mg²⁺, 200 umol dNTPs, 0.75units AmpliTaq, 50 μl final volume) were programmed using ˜0.1 ug of N.meningitidis B chromosomal DNA. Separate reactions were programmed usingDNA from N. meningitidis type B strains H4476, 250338, and BZ198.Amplification of target sequences was achieved using a standard32-cycle, three-step thermal profile, i.e. 95° C., 30 sec; 60° C., 45sec, 72° C., 1 min. Amplification was carried out in 0.2 mlpolypropylene thin-walled PCR tubes (Perkin-Elmer) in a Perkin-Elmermodel 2400 thermal cycler. All three reactions produced theNMASP-specific ˜1.4 Kbp amplimer.

The ˜1.4 Kbp NMASP amplimer was purified from unincorporated primersusing hydroxyapatite spin columns (QiaGen) and digested to completionwith an excess of NcoI and EcoRI (BRL, ˜10 units per 1 ug DNA). Thepurified and digested rNMASP ORF was then purified as described aboveand cloned into the commercially available expression plasmid pTrcHisBthat had been previously digested with both NcoI and EcoRI and treatedwith calf intestinal phosphatase to prevent vector religation (5:1,insert:vector ratio). Aliquots from the ligation reaction were then usedto transform a suitable E. coli host (e.g. TOP10) to ampicillinresistance. Mini-prep DNA from ampicillin-resistant transformants pickedat random were prepared using commercially available reagents (QiaGenMini Prep Kit) and examined for the presence of recombinant plasmidslarger than the ˜4.4 Kbp vector plasmid pTrcHis (i.e. insert-carryingplasmids). Large recombinant plasmids were then digested to completionwith NcoI and EcoRI and examined for the presence of the ˜1.4 KbpNMASP-specific fragment by standard agarose gel electrophoresis. All˜5.8 Kbp plasmids tested were found to contain the NMASP insert. PlasmidpNmAH116 was one recombinant derivative isolated by these procedures. Amap of plasmid pNmAH116 is depicted in FIG. 1.

Alternatively, to produce high levels of recombinant N. meningitidis BDegP-like protein for immunogenicity and protective efficacy studies,the NMASP ORF was PCR cloned into an E. coli high expression vectorunder the control of the stringent ara promoter (p_(BAD)).Oligonucleotide PCR primers complementary to DNA sequences encodingN-terminal amino acid residues 24 to 31 of Seq ID NO: 11 and the last 9C-terminal amino acid residues of Seq ID NO: 11 of the N. meningitidis ANMASP ORF present in the Sanger database were synthesized. In additionto the NMASP specific sequences, these PCR primers were designed tocontain flanking NcoI and XbaI restriction sites in an effort toexpedite cloning of the ORF into the commercially available expressionvector pBAD/gIII. These oligonucleotides were used to amplify aNMASP-specific PCR product from the clinically relevant N. meningitidisB strain H44/76.

The amplification primers used in these PCR reactions were designatedNMASP-G3-F-Nco (42 mer, forward primer) and NMASP-G3-RCf-Xba (47 mer,reverse primer). In addition to the NMASP coding sequence, the forwardprimer was designed to contain a unique NcoI restriction site optimallylocated upstream of the Ala₂₄ residue. Similarly, the reverse primer wasdesigned to contain an XbaI restriction site immediately downstream ofthe last NMASP codon (CAA, Q). The 3′ XbaI restriction site wasengineered into the primer such that the NMASP coding sequence would befused in frame to a myc antibody detection domain and a C-terminal (His)6 affinity purification tag encoded on the pBAD/gIII (InvitroGen) vectorplasmid.

NMASP-G3-F-Nco (SEQ ID NO: 14) 5′ - ATT ACG CAG AGG ACC ATG G CC GGC AGCTTT TTC GGT GCG GAC - 3′   42 mer NMASP-G3-RCf-Xba (SEQ ID NO: 15) 5′ -ATT ACG CAG AGG TTC TAG ACC TTG CAG GTT TAA TGC GAT AAA CAG CG - 3′   47 mer

Standard PCR amplification reactions (2 mM Mg²⁺, 200 umol dNTPs, 0.75units AmpliTaq, 50 ul final volume) were programmed using ˜0.1 ug of N.meningitidis B H44/76 chromosomal DNA. Amplification of the NMASP targetsequence was achieved using a standard 32-cycle, three-step thermalprofile, i.e. 95° C., 30 sec; 60° C., 45 sec, 72° C., 1 min.Amplification was carried out in 0.2 ml polypropylene thin-walled PCRtubes (Perkin-Elmer) in a Perkin-Elmer model 2400 thermal cycler. PCRreactions produced the predicted NMASP-specific ˜1.3 Kbp amplimer.

The ˜1.3 Kbp NMASP PCR product was purified from unincorporated primersusing hydroxyapatite spin columns (QiaGen) and digested to completionwith an excess of NcoI and XbaI (BRL, ˜10 units per 1 ug DNA) accordingto the manufacturers recommendations. The purified and digested rNMASPORF was then purified as described above and cloned into thecommercially available expression plasmid pBAD/gIII that had beenpreviously digested to completion with both NcoI and XbaI and treatedwith calf intestinal alkaline phosphatase (CIAP, BRL, ˜0.05 units/pmole5′ ends) to prevent vector religation (˜5:1, insert:vector ratio).Aliquots from the ligation reaction were then used to electrotransform asuitable E. coli host (e.g. TOP10, InvitroGen). Transformed cells wereplated on 2X-YT agar plates containing 100 ug/ml ampicillin and culturedfor ˜12-18 hours at 37° C. Mini-prep DNA from ampicillin-resistanttransformants picked at random were prepared using commerciallyavailable reagents (QiaGen Mini Prep Kit) and examined for the presenceof recombinant plasmids larger than the ˜4.1 Kbp vector plasmidpBAD/gIII (i.e. insert-carrying plasmids). These putativeinsert-carrying recombinant plasmids were then digested to completionwith NcoI and XbaI and examined for the presence of the ˜1.3 KbpNMASP-specific fragment by standard agarose gel electrophoresis (0.8%agarose, TAE buffer). All ˜5.4 Kbp plasmids tested were found to containthe NMASP insert. Plasmid pNmAH145 was one recombinant derivativeisolated by these procedures.

Expression of Recombinant NMASP Protein

The ability of pNmAH145 to express the N. meningitidis B recombinantNMASP protein was assessed by SDS-PAGE. A 5.0 ml overnight culture ofTOP10 (pNmAH145) was prepared in LB broth containing ampicillin (100ug/ml) and inoculated with cells from a “patch” plate made directly fromthe original pNmAH145 transformant colony and grown overnight at 37° C.with shaking (˜250 rpm). An aliquot of the overnight seed culture (˜1.0ml) was inoculated into a 125 ml erlenmeyer flask containing ˜25 ofLB/Ap¹⁰⁰ broth and grown at 37° C. with shaking (˜250 rpm) until theculture turbidity reached O.D.600 of ˜0.5, i.e. mid-log phase (usuallyabout 1.5-2.0 hours). At this time, approximately half of the culture(˜12.5 ml) was transferred to a second 125 ml erylenmeyer flask andexpression of recombinant NMASP protein induced by the addition ofarabinose (2.0% stock prepared in sterile water, Sigma) to a finalconcentration of 0.2%. Incubation of both the ara-induced andnon-induced cultures continued for an additional ˜4 hours at 37° C. withshaking.

Samples (˜1.0 ml) of both induced and non-induced cultures were removedfollowing the induction period and cells collected by centrifugation ina microcentrifuge (13 k×g; Eppendorf) at room temperature for ˜3-5minutes. Individual cell pellets were suspended in ˜50 ul of sterilewater, then mixed with an equal volume of 2×Lamelli SDS-PAGE samplebuffer containing 2-mercaptoethanol, and placed in boiling water bathfor ˜3-5 min to denature and reduce the recombinant protein. Equalvolumes (˜15 ul) of both the arabinose-induced and the non-induced celllysates were loaded onto duplicate 4-20% Tris/glycine polyacrylamidegradient gels (1 mm thick Mini-gels, Novex). The induced and non-inducedlysate samples were electrophoresed together with prestained molecularweight markers (SeeBlue, Novex) under conventional electrophoresisconditions (˜30 mA, constant current) using a standard SDS/Tris/glycinerunning buffer (BioRad).

Following electrophoresis, one gel was stained with commassie brilliantblue R250 (BioRad) and then destained using an aceticacid:methanol:water destaining solution to visualize novel ˜50 kDa NMASParabinose-inducible protein. The second gel was electroblotted onto aPVDF membrane (0.45 micron pore size, Novex) for ˜2 hrs at 4° C. and˜125 mA constant current using a BioRad Mini-Protean II blottingapparatus and Towbin's methanol-based (20%) transfer buffer. Blocking ofthe membrane and antibody incubations were performed using a Tris (50mM,pH7.3):CaCl₂ (1 mM):TWEEN-20™polyoxyethenlenesorbican monolaureate(0.2%) buffer containing 0.5% casein. A monoclonal anti-(His)₅ antibodyconjugated to HRP (QiaGen) was used at a {fraction (1/5,000)} dilutionto confirm the expression and identify of ˜50 kDa inducible rNMASPprotein. Visualization of the antibody reactive pattern was achieved onHyperfilm using the Amersham ECL chemiluminescence system. The resultsfrom this experiment are shown in FIG. 2.

Purification of Recombinant Protein

Recombinant NMASP protein is purified to homogeneity using standardpreparative column chromatographic procedures. Briefly, an E. colistrain harboring the expression plasmid pNmAH116 or pNmAH145 is grown inLuria broth in a 51 fermenter (New Brunswick) at 37° C. with moderateaeration until mid-log phase (˜0.5 O.D.₆₀₀) and induced with IPTG (1 mmfinal) for 4-5 hours. Cell paste is collected, washed in PBS and storedat −20° C. Aliquots of frozen cell paste (˜9-10 g wet weight) aresuspended in ˜120 ml of D-PBS by mechanical agitation and lysed bypassage through a French pressure cell (2×, 14,000 psi, 4° C.). Theexact sample preparation methodology to be used for NMASP purificationvaries somewhat depending on whether the NMASP protein is expressed as asoluble component or as insoluble inclusion bodies.

The general process for the purification of NMASP protein as a solubleprotein is given below. Insoluble material is removed after French pressdisruption by high speed centrifugation (˜10,000 ×g, 4° C., 30 min). Thesoluble fraction containing NMASP is suspended in ˜20 ml of ice cold 50mM Tris-HCl buffer (pH8.0) and loaded onto a DEAE-Sephacel SEPHACAL™(cellulose) (Pharmacia) ionic exchange column (˜5 cm×60 cm). Tominimize autoproteolysis of the NMASP protein, chromatography isconducted at 4° C. Unbound material is washed from the column usingloading buffer (50 mM Tris-Hcl, pH8.0) prior to elution of bound NMASPprotein. Elution of NMASP from the SEPHACAL ™(cellulose) matrix isachieved using a NaCl gradient (0.05-0.5 M NaCl, in 50 mM Tris-Hcl,pH8.0). Fractions released by the salt gradient are collected andexamined by standard SDS-gel electrophoresis methodologies for thepresence of a ˜40-55 kd protein. Fractions are also assayed for proteaseactivity using a standard azocasein colorimetric assay. Fractionscontaining NMASP are pooled and extensively dialyzed against 10 mMsodium phosphate buffer (SPB, pH8.0) at 4° C.

The partially purified NMASP is then applied to a hydroxylapatitecolumn, previously equilibrated in SPB. Bound proteins are eluted usinga 0.1-0.5M NaCl gradient in SPB. Fractions are collected periodicallyduring elution and examined for the presence of NMASP by SDS-gelelectrophoresis and protease activity as above. Eluted material isdialyzed against 50 mM Tris-HCl to remove residual salt and concentratedusing a Centricon-30 concentrator (Amicon, 30,000 MWCO).

Generation of a Radiolabelled Screening Probe

The sequence information shown above is used to design a pair ofnondegenerate convergent (i.e. one forward and one reverse primer)oligonucleotide NMASP-specific primers. PCR amplification of DNAfragments is performed under the same conditions as described above withthe exception that the annealing temperature is raised to 50° C. The DNAfragment is isolated from an agarose gel as before and radiolabelledusing [32P]-gamma-ATP and T4 polynucleotide kinase according to standardmethods. Unincorporated radiolabel is separated from the probe on a G25Sepharose spin column. Before use, the probe is denatured for 2 min. at95° C. and subsequently chilled on ice (4° C.).

Hybridization of Plaque-lift Filters and Southern Blots withRadiolabelled Probe

Phage plaques from library platings are immobilized on nylon filtersusing standard transfer protocols well known to those skilled in theart. Digested bacterial genomic DNA, phage or plasmid DNA iselectrophoresed on 0.8% TAE-agarose gels and transferred onto nylonfilters using a pressure blotter (Stratagene) according to themanufacturer's recommendations. Hybridizations with selected probes areperformed at 37° C. Hybridizations with other probes are generallycarried out at 60° C. Washes of increasing stringency are done at therespective hybridization temperatures until nonspecific background isminimized.

Construction of a Neisseria Meningitidis Genomic DNA Library

A genomic library is constructed in the λZAPII replacement vectorobtained from Stratgene. The vector arms are digested with EcoR1.Digests of Neisseria meningitidis DNA by EcoR1 are performed to yieldfragment sizes between 1 kb and 5 kb. Ligations of vector arms andinsert DNA are carried out according to standard protocols. Ligationreactions are packaged in vitro using the Stratagene GigaPack Gold IIIextract. The packaged phage are plated on E. coli X1 Blue MRA (P2)(Stratagene). An initial library titer is determined and expressed asnumber of pfu.

The library is screened using 4×10⁴ pfu that are plated at a density of8×10³ pfu/130 mm plate. Several putative positive phage plaques areidentified by screening the library with a radiolabelled NMASP-specificDNA hybridization probe or a NMASP-monospecific antibody and thestrongest hybridizing phage are eluted from cored agarose plugs, titeredand replated for secondary screening. The selected phages are replatedat low density (approximately 100 pfu/plate) and plaques are analyzed byPCR using primer pairs. Inserts carrying plasmids (phagemids) arerescued from the selected phage by co-infecting E. coli cells with anappropriate helper virus.

Determination of Insert Size and Mapping of DNA Fragments

In order to estimate the size of inserts, phagemid DNA is digested withNotI and the digests are analyzed on a 0.5% TAE-agarose gel side by sidewith suitable DNA markers. In order to map restriction fragments thatwould hybridize to the probe, DNA from phagemid isolates is digestedwith a number of common restriction enzymes either alone or incombination with NotI. The rationale of this approach is to discriminatebetween fragments that span the insert/phagemid vector junction andthose that map on the NotI insert. The series of single and doubledigests are run side-by-side for each phage isolate and analyzed bySouthern analysis with radiolabelled probe.

EXAMPLE: SEQUENCING OF THE NMASP NUCLEIC ACID

Sequencing of the NMASP nucleic acid from pNMASP-3 is performed usingthe plasmid pNMASP as a template. All sequencing reactions are wereperformed using the Dye Terminator Cycle Sequencing Kit fromPerkin-Elmer according to the manufacturer's specifications. Thesequencing reactions, are read using an ABI Prism 310 Genetic Analyzer.The sequences, are aligned using the AutoAssembler software(Perkin-Elmer) provided with the ABI Prism 310 sequencer. This plasmidwas inserted into E. coli Top10 (Invitrogen) and deposited with AmericanType Culture Collection (ATCC) as E. coli Top10 (pNMAH116).

EXAMPLE: GENETIC ANALYSIS Knock-out Mutants

A genomic knock-out mutation of the NMASP gene is constructed usingstandard methodologies. For example, the NMASP encoding nucleic acidfrom strain H44/76 which has been cloned into a suitable plasmid vector,e.g., plasmid pNMAH116, is digested with a restriction enzyme (e.g.,PuuII, BssIII or Asc I) that cuts the NMASP gene only once. The digestedNMASP plasmid is then ligated to a DNA fragment encoding a suitableresistance marker, e.g., the kanamycin resistance (KAN^(R)) cassettefrom plasmid pUC4-K. The ligation mixture is then used to transform E.coli cells to KAN^(R). Once the presence of the KAN^(R)-insert isconfirmed by restriction analysis, these cloned NMASPKAN^(R)-derivatives are used to transform competent N. meningitidis.Although N. meningitidis are naturally competent, standard proceduresare used to enhance transformation efficiency. Transformatnats areanalyzed by Southern blotting and/or PCR to identify knock-out mutantsthat have recombined the NMASP-KAN^(R) cassette into the chromosome.

PCR Analysis

DNA from KAN^(R) Neisseria meningitidis colonies is analyzed by PCRusing primers that hybridize to flanking sequences upstream anddownstream of the NMASP gene. A PCR product equal in size to the nativegene is only to be expected if the incoming targeting cassette has notbeen integrated into the genome by homologous recombination.Amplification products longer than the native gene are obtained onlywhen the KanR cassette has been successfully integrated.

Southern Analysis of NMASP

Genomic DNA from wild-type Neisseria meningitidis and from PCR positivedeletion mutants is digested with EcoRI. The digests are separated on a0.8% TAE-agarose gel and transferred to nylon membranes using standardprotocols. The blots are hybridized with ³²P labeled probes preparedfrom either the NMASP region or from the Kanamycin resistance gene ofpNMAH116. Using the NMASP probe, fragments having appropriate sizes aredetected in the EcoRI digests on DNA from all wild-type strains tested,whereas DNA fragments roughly 1.2 kbp longer are detected in digests onDNA from the knockout mutants. The presence of this unique, newrestriction fragment demonstrates the successful targeting of the NMASPlocus.

Probing of the membrane with the kanamycin gene does not generate anysignal in Neisseria meningitidis wild-type DNA. In DNA from the knockoutmutants, the kanamycin probe detects fragments having appropriate sizesin EcoRI digests. The presence of these sequences in the deletionmutants and their absence in the wild-type DNA demonstrates that theNMASP locus is successfully altered.

EXAMPLE: GENERATION AND REACTIVITY OF MONOCLONAL ANTI-NMASP ANTIBODIES

BALB/c mice are immunized with total outer membranes from Neisseriameningitidis or with NMASP. Hybridomas for monoclonal antibodies areprepared by fusing the spleen cells from these mice to SP2/0 cells andselecting for successful hybrids with HAT containing media. Reactivehybridomas are screened using an ELISA containing detergent extracts ofthe total outer member of Neisseria meningitidis. From this screen,hybridomas with varying levels of activity in the ELISA are selected forclonal selection, the monoclonal antibodies are assayed for reactivityto purified NMASP and total outer membranes from Neisseria meningitidisby ELISA. Monoclonal antibodies are selected that react specifically toNMASP in the ELISA.

Western blots are performed as described in Example 6.4., usingmonoclonal antibodies.

DEPOSIT OF MICROORGANISM

E. coli Top10 containing plasmid NmAH116 (pNmAH116), was deposited onAug. 21, 1998 with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas Va., 20110-2209, USA, under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedures, andassigned accession No. 98839.

The present invention is not to be limited in scope by the microorganismdeposited or the specific embodiments described herein. It will beunderstood that variations which are functionally equivalent are withinthe scope of this invention. Indeed, various modifications of theinvention, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS:  20 <210> SEQ ID NO 1 <211> LENGTH: 1347<212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis <220> FEATURE:<221> NAME/KEY: modified_base <222> LOCATION: (499)<223> OTHER INFORMATION: n=a, c, g, or t <400> SEQUENCE: 1atgctgctgc ccgactttgt ccaactggtt caaagcgaag gcccggcagt cg#tcaatatt     60caggcagccc ccgccccgcg cacccaaaac ggcagcagca atgccgaaac cg#attccgac    120ccgcttgccg acagcgaccc gttctacgaa tttttcaaac gcctcgtccc ga#acatgccc    180gaaatccccc aagaagaagc agatgacggc ggattgaact tcggttcggg ct#tcatcatc    240agcaaagacg gctatattct gaccaatacg cacgtcgtta ccggcatggg ca#gtatcaaa    300gtcctgctca acgacaagcg cgaatatacc gccaaactca tcggttcgga tg#tccaatcc    360gatgtcgccc ttctgaaaat cgacgcaacg gaagagctgc ccgtcgtcaa aa#tcggcaat    420cccaaagatt tgaaaccggg cgaatgggtc gccgccatcg gcgcgccctt cg#gcttcgac    480aacagcgtga ccgccggcnt cgtgtccgcc aaaggcagaa gcctgcccaa cg#aaagctac    540acacccttca tccaaaccga cgttgccatc aatccgggca actccggcgg cc#cgctgttc    600aacttaaaag gacaggtcgt cggcatcaac tcgcaaatat acagccgcag cg#gcggattc    660atgggcattt ccttcgccat cccgattgac gttgccatga atgtcgccga ac#agctgaaa    720aacaccggca aagtccaacg cggacaactg ggcgtgatta ttcaagaagt at#cctacggt    780ttggcacaat cgttcggttt ggacaaagcc ggcggcgcac tgattgccaa aa#tcctgccc    840ggcagccccg cagaacgtgc cggcctgcgg gcgggcgaca tcgtcctcag cc#tcgacggc    900ggagaaatac gttcttccgg cgaccttccc gttatggtcg gcgccattac gc#cgggaaaa    960gaagtcagcc tcggcgtatg gcgcaaaggc gaagaaatca caatcaaagt ca#agctgggc   1020aacgccgccg agcatatcgg cgcatcatcc aaaacagatg aagcccccta ca#ccgaacag   1080caatccggta cgttctcggt cgaatccgca ggcattaccc ttcagacaca ta#ccgacagc   1140agcggcggac acctcgtcgt cgtacgggtt tccgacgcgg cagaacgcgc ag#gcttgagg   1200cgcggcgacg aaattcttgc cgtcgggcaa gtccccgtca atgacgaagc cg#gtttccgc   1260aaagctatgg acaaggcagg caaaaacgtc cccctgctga tcatgcgccg tg#gcaacacg   1320 ctgtttatcg cattaaacct gcaataa          #                   #           1347 <210> SEQ ID NO 2 <211> LENGTH: 447<212> TYPE: PRT <213> ORGANISM: Neisseria spp. <400> SEQUENCE: 2Met Leu Leu Pro Asp Phe Val Gln Leu Val Gl #n Ser Glu Gly Pro Ala1               5    #                10   #                15Val Val Asn Ile Gln Ala Ala Pro Ala Pro Ar #g Thr Gln Asn Gly Ser            20       #            25       #            30Ser Asn Ala Glu Thr Asp Ser Asp Pro Leu Al #a Asp Ser Asp Pro Phe        35           #        40           #        45Tyr Glu Phe Phe Lys Arg Leu Val Pro Asn Me #t Pro Glu Ile Pro Gln    50               #    55               #    60Glu Glu Ala Asp Asp Gly Gly Leu Asn Phe Gl #y Ser Gly Phe Ile Ile65                   #70                   #75                   #80Ser Lys Asp Gly Tyr Ile Leu Thr Asn Thr Hi #s Val Val Thr Gly Met                85   #                90   #                95Gly Ser Ile Lys Val Leu Leu Asn Asp Lys Ar #g Glu Tyr Thr Ala Lys            100       #           105       #           110Leu Ile Gly Ser Asp Val Gln Ser Asp Val Al #a Leu Leu Lys Ile Asp        115           #       120           #       125Ala Thr Glu Glu Leu Pro Val Val Lys Ile Gl #y Asn Pro Lys Asp Leu    130               #   135               #   140Lys Pro Gly Glu Trp Val Ala Ala Ile Gly Al #a Pro Phe Gly Phe Asp145                 1 #50                 1 #55                 1 #60Asn Ser Val Thr Ala Gly Val Ser Ala Lys Gl #y Arg Ser Leu Pro Asn                165   #               170   #               175Glu Ser Tyr Thr Pro Phe Ile Gln Thr Asp Va #l Ala Ile Asn Pro Gly            180       #           185       #           190Asn Ser Gly Gly Pro Leu Phe Asn Leu Lys Gl #y Gln Val Val Gly Ile        195           #       200           #       205Asn Ser Gln Ile Tyr Ser Arg Ser Gly Gly Ph #e Met Gly Ile Ser Phe    210               #   215               #   220Ala Ile Pro Ile Asp Val Ala Met Asn Val Al #a Glu Gln Leu Lys Asn225                 2 #30                 2 #35                 2 #40Thr Gly Lys Val Gln Arg Gly Gln Leu Gly Va #l Ile Ile Gln Glu Val                245   #               250   #               255Ser Tyr Gly Leu Ala Gln Ser Phe Gly Leu As #p Lys Ala Gly Gly Ala            260       #           265       #           270Leu Ile Ala Lys Ile Leu Pro Gly Ser Pro Al #a Glu Arg Ala Gly Leu        275           #       280           #       285Arg Ala Gly Asp Ile Val Leu Ser Leu Asp Gl #y Gly Glu Ile Arg Ser    290               #   295               #   300Ser Gly Asp Leu Pro Val Met Val Gly Ala Il #e Thr Pro Gly Lys Glu305                 3 #10                 3 #15                 3 #20Val Ser Leu Gly Val Trp Arg Lys Gly Glu Gl #u Ile Thr Ile Lys Val                325   #               330   #               335Lys Leu Gly Asn Ala Ala Glu His Ile Gly Al #a Ser Ser Lys Thr Asp            340       #           345       #           350Glu Ala Pro Tyr Thr Glu Gln Gln Ser Gly Th #r Phe Ser Val Glu Ser        355           #       360           #       365Ala Gly Ile Thr Leu Gln Thr His Thr Asp Se #r Ser Gly Gly His Leu    370               #   375               #   380Val Val Val Arg Val Ser Asp Ala Ala Glu Ar #g Ala Gly Leu Arg Arg385                 3 #90                 3 #95                 4 #00Gly Asp Glu Ile Leu Ala Val Gly Gln Val Pr #o Val Asn Asp Glu Ala                405   #               410   #               415Gly Phe Arg Lys Ala Met Asp Lys Ala Gly Ly #s Asn Val Pro Leu Leu            420       #           425       #           430Ile Met Arg Arg Gly Asn Thr Leu Phe Ile Al #a Leu Asn Leu Gln        435           #       440           #       445<210> SEQ ID NO 3 <211> LENGTH: 49 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 3aagggcccaa ttacgcagag ccatggtgct gcccgacttt gtccaactg  #               49 <210> SEQ ID NO 4 <211> LENGTH: 54 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 4aagggcccaa ttacgcagag ggaattctta ttgcaggttt aatgcgataa ac#ag           54 <210> SEQ ID NO 5 <211> LENGTH: 6 <212> TYPE: PRT<213> ORGANISM: Neisseria meningitidis <400> SEQUENCE: 5Leu Thr Asn Thr His Val   1               5 <210> SEQ ID NO 6<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis<400> SEQUENCE: 6 Ser Asp Val Ala Leu   1               5<210> SEQ ID NO 7 <211> LENGTH: 7 <212> TYPE: PRT<213> ORGANISM: Neisseria meningitidis <400> SEQUENCE: 7Gly Asn Ser Gly Gly Pro Leu   1               5 <210> SEQ ID NO 8<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 8atgctgctgc ccgactttgt ccaagttcaa          #                  #           30 <210> SEQ ID NO 9 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 9 gaagcccgaa ccgaagttca atccgccgtc         #                   #           30 <210> SEQ ID NO 10 <211> LENGTH: 1395<212> TYPE: DNA <213> ORGANISM: Neisseria meningitidis<400> SEQUENCE: 10gtgttcaaaa aataccaata cttcgctttg gcggcactgt gtgccgcctt gc#tggcaggc     60tgcgaaaagg ccggcagctt tttcggtgcg gacaaaaaag aagcatcctt cg#tagaacgc    120atcgaacaca ccaaagacga cggcagtgtc agtatgctgc tgcccgactt tg#cccaactg    180gttcaaagcg aaggcccggc agtcgtcaat attcaggcag cccccgcccc gc#gcacccaa    240aacggcagcg gcaatgccga aaccgattcc gacccgcttg ccgacagcga cc#cgttctac    300gaatttttca aacgcctcgt cccgaacatg cccgaaatcc cccaagaaga ag#cagatgac    360ggcggattga acttcggttc gggcttcatc atcagcaaaa acggctacat cc#tgaccaat    420acccacgtcg ttgccggtat gggcagtatc aaagtcctgc tcaacgacaa gc#gcgaatat    480accgccaaac tcatcggttc ggatgtccaa tccgatgtcg cccttctgaa aa#tcgacgca    540acggaagagc tacccgtcgt caaaatcggc aatcccaaaa atttgaaacc gg#gcgaatgg    600gtcgctgcca tcggcgcgcc cttcggcttt gacaacagcg tgaccgccgg ca#tcgtgtcc    660gccaaaggca gaagcctgcc caacgaaagc tacacaccct tcatccaaac cg#acgttgcc    720atcaatccgg gcaattccgg cggcccgctg ttcaacttaa aaggacaggt cg#tcggcatc    780aattcgcaaa tatacagccg cagcggcgga ttcatgggca tctcctttgc ca#tcccgatt    840gacgttgcca tgaatgtcgc cgaacagctg aaaaacaccg gcaaagtcca ac#gcggacaa    900ctgggcgtga ttattcagga agtatcctac ggtttggcac agtcgttcgg tc#tggataaa    960gccagcggcg cattgattgc caaaatcctt cccggcagcc ccgcagaacg tg#ccggcctg   1020caggcgggcg acatcgtcct cagcctcgac ggcggagaaa tacgttcttc cg#gcgacctt   1080cccgtcatgg tcggcgccat tacgccggga aaagaagtca gcctcggcgt at#ggcgcaaa   1140ggcgaagaaa tcacaatcaa agccaagctg ggcaacgccg ccgagcatac cg#gcgcatca   1200tccaaaacag atgaagcccc ctacaccgaa cagcaatccg gtacgttctc gg#tcgaatcc   1260gcaggcatta cccttcagac acataccgac agcagcggca aacacctcgt cg#tcgtacgg   1320gtttccgacg cggcagaacg cgcaggctta aggcacggcg acgaaatcct ag#ccgtcagg   1380 gcaagtcccc gtcaa               #                  #                   #  1395 <210> SEQ ID NO 11 <211> LENGTH: 498<212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis<400> SEQUENCE: 11 Val Phe Lys Lys Tyr Gln Tyr Leu Ala Leu Al#a Ala Leu Cys Ala Ala   1               5  #                 10 #                 15 Ser Leu Ala Gly Cys Asp Lys Ala Gly Ser Ph#e Phe Gly Ala Asp Lys              20      #             25     #             30 Lys Glu Ala Ser Phe Val Glu Arg Ile Lys Hi#s Thr Lys Asp Asp Gly          35          #         40         #         45 Ser Val Ser Met Leu Leu Pro Asp Phe Val Gl#n Leu Val Gln Ser Glu      50              #     55             #     60 Gly Pro Ala Val Val Asn Ile Gln Ala Ala Pr#o Ala Pro Arg Thr Gln  65                  # 70                 # 75                  # 80 Asn Gly Ser Ser Asn Ala Glu Thr Asp Ser As#p Pro Leu Ala Asp Ser                  85  #                 90 #                 95 Asp Pro Phe Tyr Glu Phe Phe Lys Arg Leu Va#l Pro Asn Met Pro Glu             100       #           105      #           110 Ile Pro Gln Glu Glu Ala Asp Asp Gly Gly Le#u Asn Phe Gly Ser Gly         115           #       120          #       125 Phe Ile Ile Ser Lys Asp Gly Tyr Ile Leu Th#r Asn Thr His Val Val     130               #   135              #   140 Thr Gly Met Gly Ser Ile Lys Val Leu Leu As#n Asp Lys Arg Glu Tyr 145                 1 #50                 1#55                 1 #60 Thr Ala Lys Leu Ile Gly Ser Asp Val Gln Se#r Asp Val Ala Leu Leu                 165   #               170  #               175 Lys Ile Asp Ala Thr Glu Glu Leu Pro Val Va#l Lys Ile Gly Asn Pro             180       #           185      #           190 Lys Asp Leu Lys Pro Gly Glu Trp Val Ala Al#a Ile Gly Ala Pro Phe         195           #       200          #       205 Gly Phe Asp Asn Ser Val Thr Ala Gly Val Se#r Ala Lys Gly Arg Ser     210               #   215              #   220 Leu Pro Asn Glu Ser Tyr Thr Pro Phe Ile Gl#n Thr Asp Val Ala Ile 225                 2 #30                 2#35                 2 #40 Asn Pro Gly Asn Ser Gly Gly Pro Leu Phe As#n Leu Lys Gly Gln Val                 245   #               250  #               255 Val Gly Ile Asn Ser Gln Ile Tyr Ser Arg Se#r Gly Gly Phe Met Gly             260       #           265      #           270 Ile Ser Phe Ala Ile Pro Ile Asp Val Ala Me#t Asn Val Ala Glu Gln         275           #       280          #       285 Leu Lys Asn Thr Gly Lys Val Gln Arg Gly Gl#n Leu Gly Val Ile Ile     290               #   295              #   300 Gln Glu Val Ser Tyr Gly Leu Ala Gln Ser Ph#e Gly Leu Asp Lys Ala 305                 3 #10                 3#15                 3 #20 Gly Gly Ala Leu Ile Ala Lys Ile Leu Pro Gl#y Ser Pro Ala Glu Arg                 325   #               330  #               335 Ala Gly Leu Arg Ala Gly Asp Ile Val Leu Se#r Leu Asp Gly Gly Glu             340       #           345      #           350 Ile Arg Ser Ser Gly Asp Leu Pro Val Met Va#l Gly Ala Ile Thr Pro         355           #       360          #       365 Gly Lys Glu Val Ser Leu Gly Val Trp Arg Ly#s Gly Glu Glu Ile Thr     370               #   375              #   380 Ile Lys Val Lys Leu Gly Asn Ala Ala Glu Hi#s Ile Gly Ala Ser Ser 385                 3 #90                 3#95                 4 #00 Lys Thr Asp Glu Ala Pro Tyr Thr Glu Gln Gl#n Ser Gly Thr Phe Ser                 405   #               410  #               415 Val Glu Ser Ala Gly Ile Thr Leu Gln Thr Hi#s Thr Asp Ser Ser Gly             420       #           425      #           430 Gly His Leu Val Val Val Arg Val Ser Asp Al#a Ala Glu Arg Ala Gly         435           #       440          #       445 Leu Arg Arg Gly Asp Glu Ile Leu Ala Val Gl#y Gln Val Pro Val Asn     450               #   455              #   460 Asp Glu Ala Gly Phe Arg Lys Ala Met Asp Ly#s Ala Gly Lys Asn Val 465                 4 #70                 4#75                 4 #80 Pro Leu Leu Ile Met Arg Arg Gly Asn Thr Le#u Phe Ile Ala Leu Asn                 485   #               490  #               495 Leu Gln <210> SEQ ID NO 12 <211> LENGTH: 475<212> TYPE: PRT <213> ORGANISM: Neisseria meningitidis<400> SEQUENCE: 12 Ala Gly Ser Phe Phe Gly Ala Asp Lys Lys Gl#u Ala Ser Phe Val Glu   1               5  #                 10 #                 15 Arg Ile Lys His Thr Lys Asp Asp Gly Ser Va#l Ser Met Leu Leu Pro              20      #             25     #             30 Asp Phe Val Gln Leu Val Gln Ser Glu Gly Pr#o Ala Val Val Asn Ile          35          #         40         #         45 Gln Ala Ala Pro Ala Pro Arg Thr Gln Asn Gl#y Ser Ser Asn Ala Glu      50              #     55             #     60 Thr Asp Ser Asp Pro Leu Ala Asp Ser Asp Pr#o Phe Tyr Glu Phe Phe  65                  # 70                 # 75                  # 80 Lys Arg Leu Val Pro Asn Met Pro Glu Ile Pr#o Gln Glu Glu Ala Asp                  85  #                 90 #                 95 Asp Gly Gly Leu Asn Phe Gly Ser Gly Phe Il#e Ile Ser Lys Asp Gly             100       #           105      #           110 Tyr Ile Leu Thr Asn Thr His Val Val Thr Gl#y Met Gly Ser Ile Lys         115           #       120          #       125 Val Leu Leu Asn Asp Lys Arg Glu Tyr Thr Al#a Lys Leu Ile Gly Ser     130               #   135              #   140 Asp Val Gln Ser Asp Val Ala Leu Leu Lys Il#e Asp Ala Thr Glu Glu 145                 1 #50                 1#55                 1 #60 Leu Pro Val Val Lys Ile Gly Asn Pro Lys As#p Leu Lys Pro Gly Glu                 165   #               170  #               175 Trp Val Ala Ala Ile Gly Ala Pro Phe Gly Ph#e Asp Asn Ser Val Thr             180       #           185      #           190 Ala Gly Val Ser Ala Lys Gly Arg Ser Leu Pr#o Asn Glu Ser Tyr Thr         195           #       200          #       205 Pro Phe Ile Gln Thr Asp Val Ala Ile Asn Pr#o Gly Asn Ser Gly Gly     210               #   215              #   220 Pro Leu Phe Asn Leu Lys Gly Gln Val Val Gl#y Ile Asn Ser Gln Ile 225                 2 #30                 2#35                 2 #40 Tyr Ser Arg Ser Gly Gly Phe Met Gly Ile Se#r Phe Ala Ile Pro Ile                 245   #               250  #               255 Asp Val Ala Met Asn Val Ala Glu Gln Leu Ly#s Asn Thr Gly Lys Val             260       #           265      #           270 Gln Arg Gly Gln Leu Gly Val Ile Ile Gln Gl#u Val Ser Tyr Gly Leu         275           #       280          #       285 Ala Gln Ser Phe Gly Leu Asp Lys Ala Gly Gl#y Ala Leu Ile Ala Lys     290               #   295              #   300 Ile Leu Pro Gly Ser Pro Ala Glu Arg Ala Gl#y Leu Arg Ala Gly Asp 305                 3 #10                 3#15                 3 #20 Ile Val Leu Ser Leu Asp Gly Gly Glu Ile Ar#g Ser Ser Gly Asp Leu                 325   #               330  #               335 Pro Val Met Val Gly Ala Ile Thr Pro Gly Ly#s Glu Val Ser Leu Gly             340       #           345      #           350 Val Trp Arg Lys Gly Glu Glu Ile Thr Ile Ly#s Val Lys Leu Gly Asn         355           #       360          #       365 Ala Ala Glu His Ile Gly Ala Ser Ser Lys Th#r Asp Glu Ala Pro Tyr     370               #   375              #   380 Thr Glu Gln Gln Ser Gly Thr Phe Ser Val Gl#u Ser Ala Gly Ile Thr 385                 3 #90                 3#95                 4 #00 Leu Gln Thr His Thr Asp Ser Ser Gly Gly Hi#s Leu Val Val Val Arg                 405   #               410  #               415 Val Ser Asp Ala Ala Glu Arg Ala Gly Leu Ar#g Arg Gly Asp Glu Ile             420       #           425      #           430 Leu Ala Val Gly Gln Val Pro Val Asn Asp Gl#u Ala Gly Phe Arg Lys         435           #       440          #       445 Ala Met Asp Lys Ala Gly Lys Asn Val Pro Le#u Leu Ile Met Arg Arg     450               #   455              #   460 Gly Asn Thr Leu Phe Ile Ala Leu Asn Leu Gl #n465                 4 #70                 4 #75 <210> SEQ ID NO 13<211> LENGTH: 1326 <212> TYPE: DNA<213> ORGANISM: Neisseria meningitidis <400> SEQUENCE: 13gccggcagct ttttcggtgc ggacaaaaaa gaagcatcct tcgtagaacg ca#tcgaacac     60accaaagacg acggcagtgt cagtatgctg ctgcccgact ttgcccaact gg#ttcaaagc    120gaaggcccgg cagtcgtcaa tattcaggca gcccccgccc cgcgcaccca aa#acggcagc    180ggcaatgccg aaaccgattc cgacccgctt gccgacagcg acccgttcta cg#aatttttc    240aaacgcctcg tcccgaacat gcccgaaatc ccccaagaag aagcagatga cg#gcggattg    300aacttcggtt cgggcttcat catcagcaaa aacggctaca tcctgaccaa ta#cccacgtc    360gttgccggta tgggcagtat caaagtcctg ctcaacgaca agcgcgaata ta#ccgccaaa    420ctcatcggtt cggatgtcca atccgatgtc gcccttctga aaatcgacgc aa#cggaagag    480ctacccgtcg tcaaaatcgg caatcccaaa aatttgaaac cgggcgaatg gg#tcgctgcc    540atcggcgcgc ccttcggctt tgacaacagc gtgaccgccg gcatcgtgtc cg#ccaaaggc    600agaagcctgc ccaacgaaag ctacacaccc ttcatccaaa ccgacgttgc ca#tcaatccg    660ggcaattccg gcggcccgct gttcaactta aaaggacagg tcgtcggcat ca#attcgcaa    720atatacagcc gcagcggcgg attcatgggc atctcctttg ccatcccgat tg#acgttgcc    780atgaatgtcg ccgaacagct gaaaaacacc ggcaaagtcc aacgcggaca ac#tgggcgtg    840attattcagg aagtatccta cggtttggca cagtcgttcg gtctggataa ag#ccagcggc    900gcattgattg ccaaaatcct tcccggcagc cccgcagaac gtgccggcct gc#aggcgggc    960gacatcgtcc tcagcctcga cggcggagaa atacgttctt ccggcgacct tc#ccgtcatg   1020gtcggcgcca ttacgccggg aaaagaagtc agcctcggcg tatggcgcaa ag#gcgaagaa   1080atcacaatca aagccaagct gggcaacgcc gccgagcata ccggcgcatc at#ccaaaaca   1140gatgaagccc cctacaccga acagcaatcc ggtacgttct cggtcgaatc cg#caggcatt   1200acccttcaga cacataccga cagcagcggc aaacacctcg tcgtcgtacg gg#tttccgac   1260gcggcagaac gcgcaggctt aaggcacggc gacgaaatcc tagccgtcag gg#caagtccc   1320 cgtcaa                  #                  #                   #         1326 <210> SEQ ID NO 14 <211> LENGTH: 42<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 14 attacgcaga ggaccatggc cggcagcttt ttcggtgcgg ac    #                   #  42 <210> SEQ ID NO 15 <211> LENGTH: 47<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 15attacgcaga ggttctagac cttgcaggtt taatgcgata aacagcg   #                47 <210> SEQ ID NO 16 <211> LENGTH: 51 <212> TYPE: PRT<213> ORGANISM: Neisseria meningitidis <400> SEQUENCE: 16Val Phe Lys Lys Tyr Gln Tyr Leu Ala Leu Al #a Ala Leu Cys Ala Ala  1               5  #                 10  #                 15Ser Leu Ala Gly Cys Asp Lys Ala Gly Ser Ph #e Phe Gly Ala Asp Lys             20      #             25      #             30Lys Glu Ala Ser Phe Val Glu Arg Ile Lys Hi #s Thr Lys Asp Asp Gly         35          #         40          #         45 Ser Val Ser     50 <210> SEQ ID NO 17 <211> LENGTH: 153 <212> TYPE: DNA<213> ORGANISM: Neisseria meningitidis <400> SEQUENCE: 17gtgttcaaaa aataccaata cctcgctttg gcagcactgt gtgccgcctc gc#tggcaggc     60tgcgacaaag ccggcagctt tttcggtgcg gacaaaaaag aagcatcctt tg#tagaacgc    120 atcaaacaca ccaaagacga cggcagcgtc agt       #                   #        153 <210> SEQ ID NO 18 <211> LENGTH: 24<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 18 gtgttcaaaa aataccaata cctc          #                   #                24 <210> SEQ ID NO 19<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of Artificial #Sequence: Primer <400> SEQUENCE: 19actgacgctg ccgtcgtctt tggt           #                  #                24 <210> SEQ ID NO 20 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence: Primer<400> SEQUENCE: 20 ttgcaggttt aatgcgataa acagcgt          #                   #             27

What is claimed is:
 1. An isolated nucleic acid comprising thenucleotide sequence encoding the polypeptide of SEQ ID NO.
 2. 2. Anisolated nucleic acid comprising the nucleotide sequence of SEQ IDNO.:
 1. 3. A pharmaceutical composition comprising the isolated nucleicacid of any one of claims 1 or
 2. 4. A vector, comprising the nucleicacid sequence of any one of claims 1 or
 2. 5. A host cell, comprising avector, said vector comprising the nucleic acid of any one of claims 1or 2.